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INT E R NAT I ONAL T AB L E S
FOR
C RYST AL L OGR APHY
International Tables for Crystallography
Volume A: Space-Group Symmetry

Editor Theo Hahn
First Edition 1983, Fourth Edition 1995
Corrected Reprint 1996
Volume B: Reciprocal Space

Editor U. Shmueli
First Edition 1993, Corrected Reprint 1996
Second Edition 2001
Volume C: Mathematical, Physical and Chemical Tables

Editors A. J. C. Wilson and E. Prince
First Edition 1992, Corrected Reprint 1995
Second Edition 1999
Volume F: Crystallography of Biological Macromolecules

Editors Michael G. Rossmann and Eddy Arnold
First Edition 2001
Forthcoming volumes
Volume D: Physical Properties of Crystals

Editor A. Authier
Volume E: Subperiodic Groups

Editors V. Kopsky and D. B. Litvin
Volume A1: Symmetry Relations between Space Groups

Editors H. Wondratschek and U. Müller
INTERNATIONAL TABLES
FOR
CRYSTALLOGRAPHY
Volume F
CRYSTALLOGRAPHY OF BIOLOGICAL MACROMOLECULES
Edited by
MICHAEL G. ROSSMANN AND EDDY ARNOLD
Published for
T HE I NT E RNAT IONAL UNION OF C RYST AL L OGR APHY
by
KL UW E R ACADE MIC PUBLISHERS
DORDRE CHT /BOST ON/L ONDON
2001
A C.I.P. Catalogue record for this book
is available from the Library of Congress
ISBN 0-7923-6857-6 (acid-free paper)
Published by Kluwer Academic Publishers,

P.O. Box 17, 3300 AA Dordrecht, The Netherlands
Sold and distributed in North, Central and South America
by Kluwer Academic Publishers,
101 Philip Drive, Norwell, MA 02061, USA
In all other countries, sold and distributed
by Kluwer Academic Publishers,
P.O. Box 322, 3300 AH Dordrecht, The Netherlands
Technical Editor: N. J. Ashcroft

# International Union of Crystallography 2001
Short extracts may be
reproduced without formality, provided that the
source is acknowledged, but substantial portions
may not be reproduced by any process
without written permission from the
International Union of Crystallography
Printed in Denmark by P. J. Schmidt A/S
Dedicated to
DAVID C. PHILLIPS
The editors wish to express their special thanks to David Phillips (Lord Phillips of
Ellesmere) and Professor Louise Johnson for contributing an exceptional chapter on
the structure determination of hen egg-white lysozyme. Although Chapter 26.1
describes the first structural investigations of an enzyme, the procedures used are still
as fresh and important today as they were 35 years ago and this chapter is strongly
recommended to students of both crystallography and enzymology. Completion of
this chapter was David’s last scientific accomplishment only a few weeks before his
death. This volume of International Tables for Crystallography is dedicated to the
memory of David C. Phillips in recognition of his pivotal contributions to the
foundations of the crystallography of biological macromolecules.
Advisors and Advisory Board
Advisors: J. Drenth, A. Liljas. Advisory Board: U. W. Arndt, E. N. Baker, S. C. Harrison, W. G. J. Hol, K. C. Holmes, L. N. Johnson,
H. M. Berman, T. L. Blundell, M. Bolognesi, A. T. Brunger, C. E. Bugg, K. K. Kannan, S.-H. Kim, A. Klug, D. Moras, R. J. Read,
R. Chandrasekaran, P. M. Colman, D. R. Davies, J. Deisenhofer, T. J. Richmond, G. E. Schulz, P. B. Sigler,² D. I. Stuart, T. Tsukihara,
R. E. Dickerson, G. G. Dodson, H. Eklund, R. GiegeÂ, J. P. Glusker, M. Vijayan, A. Yonath.
Contributing authors
E. E. Abola: The Department of Molecular Biology, The Scripps Research W. Chiu: Verna and Marrs McLean Department of Biochemistry and Molecular
Institute, La Jolla, CA 92037, USA. [24.1] Biology, Baylor College of Medicine, Houston, Texas 77030, USA. [19.2]
P. D. Adams: The Howard Hughes Medical Institute and Department of Molecular J. C. Cole: Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA. CB2 1EZ, England. [22.4]
[18.2, 25.2.3] M. L. Connolly: 1259 El Camino Real #184, Menlo Park, CA 94025, USA.
F. H. Allen: Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge [22.1.2]
CB2 1EZ, England. [22.4, 24.3]
K. D. Cowtan: Department of Chemistry, University of York, York YO1 5DD,
U. W. Arndt: Laboratory of Molecular Biology, Medical Research Council, Hills England. [15.1, 25.2.2]
Road, Cambridge CB2 2QH, England. [6.1]
D. W. J. Cruickshank: Chemistry Department, UMIST, Manchester M60 1QD,
E. Arnold: Biomolecular Crystallography Laboratory, Center for Advanced England.†† [18.5]
Biotechnology and Medicine & Rutgers University, 679 Hoes Lane, Piscat-
away, NJ 08854-5638, USA. [1.1, 1.4.1, 13.4, 25.1] V. M. Dadarlat: Department of Medicinal Chemistry and Molecular Pharma-
cology, Purdue University, West Lafayette, Indiana 47907-1333, USA. [20.2]
E. N. Baker: School of Biological Sciences, University of Auckland, Private Bag
92-109, Auckland, New Zealand. [22.2] U. Das: Unité de Conformation de Macromolécules Biologiques, Université Libre
de Bruxelles, avenue F. D. Roosevelt 50, CP160/16, B-1050 Bruxelles,
T. S. Baker: Department of Biological Sciences, Purdue University, West Lafay- Belgium. [21.2]
ette, Indiana 47907-1392, USA. [19.6]
Z. Dauter: National Cancer Institute, Brookhaven National Laboratory, Building
C. G. van Beek: Department of Biological Sciences, Purdue University, West 725A-X9, Upton, NY 11973, USA. [9.1, 18.4]
Lafayette, IN 47907-1392, USA.‡ [11.5]
D. R. Davies: Laboratory of Molecular Biology, National Institute of Diabetes and
J. Berendzen: Biophysics Group, Mail Stop D454, Los Alamos National Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
Laboratory, Los Alamos, NM 87545, USA. [14.2.2] 20892-0560, USA. [4.3]
H. M. Berman: The Nucleic Acid Database Project, Department of Chemistry, W. L. DeLano: Graduate Group in Biophysics, Box 0448, University of California,
Rutgers University, 610 Taylor Road, Piscataway, NJ 08854-8077, USA. [21.2, San Francisco, CA 94143, USA. [25.2.3]
24.2, 24.5]
R. E. Dickerson: Molecular Biology Institute, University of California, Los
T. N. Bhat: National Institute of Standards and Technology, Biotechnology Angeles, Los Angeles, CA 90095-1570, USA. [23.3]
Division, 100 Bureau Drive, Gaithersburg, MD 20899, USA. [24.5]
J. Ding: Biomolecular Crystallography Laboratory, CABM & Rutgers University,
C. C. F. Blake: Davy Faraday Research Laboratory, The Royal Institution, London
679 Hoes Lane, Piscataway, NJ 08854-5638, USA, and Institute of Biochem-
W1X 4BS, England.§ [26.1]
istry and Cell Biology, Chinese Academy of Sciences, Yue-Yang Road,
D. M. Blow: Biophysics Group, Blackett Laboratory, Imperial College of Science, Shanghai 200 031, People’s Republic of China. [25.1]
Technology & Medicine, London SW7 2BW, England. [13.1]
J. Drenth: Laboratory of Biophysical Chemistry, University of Groningen,
T. L. Blundell: Department of Biochemistry, University of Cambridge, 80 Tennis Nijenborgh 4, 9747 AG Groningen, The Netherlands. [2.1]
Court Road, Cambridge CB2 1GA, England. [12.1]
O. Dym: UCLA–DOE Laboratory of Structural Biology and Molecular Medicine,
R. Bolotovsky: Department of Biological Sciences, Purdue University, West UCLA, Box 951570, Los Angeles, CA 90095-1570, USA. [21.3]
Lafayette, IN 47907-1392, USA.} [11.5]
E. F. Eikenberry: Swiss Light Source, Paul Scherrer Institut, 5232 Villigen PSI,
P. E. Bourne: Department of Pharmacology, University of California, San Diego, Switzerland. [7.1, 7.2]
9500 Gilman Drive, La Jolla, CA 92093-0537, USA. [24.5]
D. Eisenberg: UCLA–DOE Laboratory of Structural Biology and Molecular
G. Bricogne: Laboratory of Molecular Biology, Medical Research Council, Medicine, Department of Chemistry & Biochemistry, Molecular Biology
Cambridge CB2 2QH, England. [16.2] Institute and Department of Biological Chemistry, UCLA, Los Angeles, CA
A. T. Brunger: Howard Hughes Medical Institute, and Departments of Molecular 90095-1570, USA. [21.3]
and Cellular Physiology, Neurology and Neurological Sciences, and Stanford D. M. Engelman: Department of Molecular Biophysics and Biochemistry, Yale
Synchrotron Radiation Laboratory (SSRL), Stanford University, 1201 Welch University, New Haven, CT 06520, USA. [19.4]
Road, MSLS P210, Stanford, CA 94305, USA. [18.2, 25.2.3]
R. A. Engh: Pharmaceutical Research, Roche Diagnostics GmbH, Max Planck
A. Burgess Hickman: Laboratory of Molecular Biology, National Institute of Institut für Biochemie, 82152 Martinsried, Germany. [18.3]
Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892-0560, USA. [4.3] Z. Feng: The Nucleic Acid Database Project, Department of Chemistry, Rutgers
University, 610 Taylor Road, Piscataway, NJ 08854-8077, USA. [24.2, 24.5]
H. L. Carrell: The Institute for Cancer Research, The Fox Chase Cancer Center,
Philadelphia, PA 19111, USA. [5.1] R. H. Fenn: Davy Faraday Research Laboratory, The Royal Institution, London
W1X 4BS, England.‡‡ [26.1]
D. Carvin: Biomolecular Modelling Laboratory, Imperial Cancer Research Fund,
44 Lincoln’s Inn Field, London WC2A 3PX, England. [12.1] W. Furey: Biocrystallography Laboratory, VA Medical Center, PO Box 12055,
University Drive C, Pittsburgh, PA 15240, USA, and Department of Pharma-
R. Chandrasekaran: Whistler Center for Carbohydrate Research, Purdue
cology, University of Pittsburgh School of Medicine, 1340 BSTWR, Pitts-
University, West Lafayette, IN 47907, USA. [19.5]
burgh, PA 15261, USA. [25.2.1]
M. S. Chapman: Department of Chemistry & Institute of Molecular Biophysics,
M. Gerstein: Department of Molecular Biophysics & Biochemistry, 266 Whitney
Florida State University, Tallahassee, FL 32306-4380, USA. [22.1.2]
Avenue, Yale University, PO Box 208114, New Haven, CT 06520, USA.
[22.1.1]
† Deceased.
‡ Present address: RJ Lee Instruments, 515 Pleasant Valley Road, Trafford, PA R. GiegeÂ: Unité Propre de Recherche du CNRS, Institut de Biologie Moléculaire et
15085, USA. Cellulaire, 15 rue René Descartes, F-67084 Strasbourg CEDEX, France. [4.1]
§ Present address: Kent House, 19 The Warren, Cromer, Norfolk NR27 0AR,
England. †† Present address: 105 Moss Lane, Alderley Edge, Cheshire SK9 7HW, England.
} Present address: Philips Analytical Inc., 12 Michigan Drive, Natick, MA 01760, ‡‡ Present address: 2 Second Avenue, Denvilles, Havant, Hampshire PO9 2QP,
USA. England.
vi
G. L. Gilliland: Center for Advanced Research in Biotechnology of the Maryland V. S. Lamzin: European Molecular Biology Laboratory (EMBL), Hamburg
Biotechnology Institute and National Institute of Standards and Technology, Outstation, c/o DESY, Notkestr. 85, 22603 Hamburg, Germany. [25.2.5]
9600 Gudelsky Dr., Rockville, MD 20850, USA. [24.4, 24.5] R. A. Laskowski: Department of Crystallography, Birkbeck College, University of
J. P. Glusker: The Institute for Cancer Research, The Fox Chase Cancer Center, London, Malet Street, London WC1E 7HX, England. [25.2.6]
Philadelphia, PA 19111, USA. [5.1]
A. G. W. Leslie: MRC Laboratory of Molecular Biology, Hills Road, Cambridge
P. Gros: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular CB2 2QH, England. [11.2]
Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
D. Lin: Biology Department, Bldg 463, Brookhaven National Laboratory, Upton,
[25.2.3]
NY 11973-5000, USA. [24.1]
R. W. Grosse-Kunstleve: The Howard Hughes Medical Institute and Department
of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT M. W. MacArthur: Biochemistry and Molecular Biology Department, University
06511, USA. [25.2.3] College London, Gower Street, London WC1E 6BT, England. [25.2.6]
S. M. Gruner: Department of Physics, 162 Clark Hall, Cornell University, Ithaca, A. McPherson: Department of Molecular Biology & Biochemistry, University of
NY 14853-2501, USA. [7.1, 7.2] California at Irvine, Irvine, CA 92717, USA. [4.1]
W. F. van Gunsteren: Laboratory of Physical Chemistry, ETH-Zentrum, 8092 P. Main: Department of Physics, University of York, York YO1 5DD, England.
Zürich, Switzerland. [20.1] [15.1, 25.2.2]
H. A. Hauptman: Hauptman–Woodward Medical Research Institute, Inc., 73 High G. A. Mair: Davy Faraday Research Laboratory, The Royal Institution, London
Street, Buffalo, NY 14203-1196, USA. [16.1] W1X 4BS, England.§ [26.1]
J. R. Helliwell: Department of Chemistry, University of Manchester, M13 9PL, N. O. Manning: Biology Department, Bldg 463, Brookhaven National Laboratory,
England. [8.1] Upton, NY 11973-5000, USA. [24.1]
R. Henderson: Medical Research Council, Laboratory of Molecular Biology, B. W. Matthews: Institute of Molecular Biology, Howard Hughes Medical Insti-
Hills Road, Cambridge CB2 2QH, England. [19.6] tute and Department of Physics, University of Oregon, Eugene, OR 97403,
W. A. Hendrickson: Department of Biochemistry, College of Physicians & USA. [14.1]
Surgeons of Columbia University, 630 West 168th Street, New York, NY C. Mattos: Department of Molecular and Structural Biochemistry, North Carolina
10032, USA. [14.2.1] State University, 128 Polk Hall, Raleigh, NC 02795, USA. [23.4]
A. E. Hodel: Department of Biochemistry, Emory University School of Medicine, H. Michel: Max-Planck-Institut für Biophysik, Heinrich-Hoffmann-Strasse 7,
Atlanta, GA 30322, USA. [23.2] D-60528 Frankfurt/Main, Germany. [4.2]
W. G. J. Hol: Biomolecular Structure Center, Department of Biological Structure, R. Miller: Hauptman–Woodward Medical Research Institute, Inc., 73 High Street,
Howard Hughes Medical Institute, University of Washington, Seattle, WA Buffalo, NY 14203-1196, USA. [16.1]
98195-7742, USA. [1.3]
W. Minor: Department of Molecular Physiology and Biological Physics, Univer-
L. Holm: EMBL–EBI, Cambridge CB10 1SD, England. [23.1.2] sity of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
H. Hope: Department of Chemistry, University of California, Davis, One Shields [11.4]
Ave, Davis, CA 95616-5295, USA. [10.1] K. Moffat: Department of Biochemistry and Molecular Biology, The Center for
V. J. Hoy: Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge Advanced Radiation Sources, and The Institute for Biophysical Dynamics, The
CB2 1EZ, England. [24.3] University of Chicago, Chicago, Illinois 60637, USA. [8.2]
R. Huber: Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany. P. B. Moore: Departments of Chemistry and Molecular Biophysics and
[12.2, 18.3] Biochemistry, Yale University, New Haven, CT 06520, USA. [19.4]
S. H. Hughes: National Cancer Institute, Frederick Cancer R&D Center, Frederick, G. N. Murshudov: Structural Biology Laboratory, Department of Chemistry,
MD 21702-1201, USA. [3.1] University of York, York YO10 5DD, England, and CLRC, Daresbury
S. A. Islam: Institute of Cancer Research, 44 Lincoln’s Inn Fields, London WC2A Laboratory, Daresbury, Warrington, WA4 4AD, England. [18.4]
3PX, England. [12.1] J. Navaza: Laboratoire de Génétique des Virus, CNRS-GIF, 1. Avenue de la
J.-S. Jiang: Biology Department, Bldg 463, Brookhaven National Laboratory, Terrasse, 91198 Gif-sur-Yvette, France. [13.2]
Upton, NY 11973-5000, USA. [24.1, 25.2.3] A. C. T. North: Davy Faraday Research Laboratory, The Royal Institution,
J. E. Johnson: Department of Molecular Biology, The Scripps Research Institute, London W1X 4BS, England.} [26.1]
10550 N. Torrey Pines Road, La Jolla, California 92037, USA. [19.3] J. W. H. Oldham.² [26.1]
L. N. Johnson: Davy Faraday Research Laboratory, The Royal Institution, London A. J. Olson: The Scripps Research Institute, La Jolla, CA 92037, USA. [17.2]
W1X 4BS, England.‡ [26.1]
C. Orengo: Biomolecular Structure and Modelling Unit, Department of
T. A. Jones: Department of Cell and Molecular Biology, Uppsala University, Biochemistry and Molecular Biology, University College, Gower Street,
Biomedical Centre, Box 596, SE-751 24 Uppsala, Sweden. [17.1] London WC1E 6BT, England. [23.1.1]
W. Kabsch: Max-Planck-Institut für medizinische Forschung, Abteilung Z. Otwinowski: UT Southwestern Medical Center at Dallas, 5323 Harry Hines
Biophysik, Jahnstrasse 29, 69120 Heidelberg, Germany. [11.3, 25.2.9] Boulevard, Dallas, TX 75390-9038, USA. [11.4]
M. Kjeldgaard: Institute of Molecular and Structural Biology, University of
N. S. Pannu: Department of Mathematical Sciences, University of Alberta,
Aarhus, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark. [17.1]
Edmonton, Alberta, Canada T6G 2G1. [25.2.3]
G. J. Kleywegt: Department of Cell and Molecular Biology, Uppsala University,
A. Perrakis: European Molecular Biology Laboratory (EMBL), Grenoble
Biomedical Centre, Box 596, SE-751 24 Uppsala, Sweden. [17.1, 21.1]
Outstation, c/o ILL, Avenue des Martyrs, BP 156, 38042 Grenoble CEDEX 9,
R. Knott: Small Angle Scattering Facility, Australian Nuclear Science & Tech- France. [25.2.5]
nology Organisation, Physics Division, PMB 1 Menai NSW 2234, Australia.
[6.2] D. C. Phillips.² [26.1]
D. F. Koenig: Davy Faraday Research Laboratory, The Royal Institution, London R. J. Poljak: Davy Faraday Research Laboratory, The Royal Institution, London
W1X 4BS, England.§ [26.1] W1X 4BS, England.†† [26.1]
A. A. Kossiakoff: Department of Biochemistry and Molecular Biology, CLSC J. Pontius: Unité de Conformation de Macromolécules Biologiques, Université
161A, University of Chicago, Chicago, IL 60637, USA. [19.1] Libre de Bruxelles, avenue F. D. Roosevelt 50, CP160/16, B-1050 Bruxelles,
Belgium. [21.2]
P. J. Kraulis: Stockholm Bioinformatics Center, Department of Biochemistry,
Stockholm University, SE-106 91 Stockholm, Sweden. [25.2.7] C. B. Post: Department of Medicinal Chemistry and Molecular Pharmacology,
Purdue University, West Lafayette, Indiana 47907-1333, USA. [20.2]
J. E. Ladner: Center for Advanced Research in Biotechnology of the Maryland
Biotechnology Institute and National Institute of Standards and Technology, J. Prilusky: Bioinformatics Unit, Weizmann Institute of Science, Rehovot 76100,
9600 Gudelsky Dr., Rockville, MD 20850, USA. [24.4] Israel. [24.1]
} Present address: Prospect House, 27 Breary Lane, Bramhope, Leeds LS16 9AD,
‡ Present address: Laboratory of Molecular Biophysics, Rex Richards Building, England.
South Parks Road, Oxford OX1 3QU, England. † Deceased.
§ Present address unknown. †† Present address: CARB, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
vii
F. A. Quiocho: Howard Hughes Medical Institute and Department of Biochem- M. W. Tate: Department of Physics, 162 Clark Hall, Cornell University, Ithaca, NY
istry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, 14853-2501, USA. [7.1, 7.2]
USA. [23.2] L. F. Ten Eyck: San Diego Supercomputer Center 0505, University of California at
R. J. Read: Department of Haematology, University of Cambridge, Wellcome San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0505, USA. [18.1, 25.2.4]
Trust Centre for Molecular Mechanisms in Disease, CIMR, Wellcome Trust/ T. C. Terwilliger: Bioscience Division, Mail Stop M888, Los Alamos National
MRC Building, Hills Road, Cambridge CB2 2XY, England. [15.2, 25.2.3] Laboratory, Los Alamos, NM 87545, USA. [14.2.2]
L. M. Rice: Department of Molecular Biophysics and Biochemistry, Yale J. M. Thornton: Biochemistry and Molecular Biology Department, University
University, New Haven, CT 06511, USA. [18.2, 25.2.3] College London, Gower Street, London WC1E 6BT, England, and Department
F. M. Richards: Department of Molecular Biophysics & Biochemistry, 266 of Crystallography, Birkbeck College, University of London, Malet Street,
Whitney Avenue, Yale University, PO Box 208114, New Haven, CT 06520, London WC1E 7HX, England. [23.1.1, 25.2.6]
USA. [22.1.1] L. Tong: Department of Biological Sciences, Columbia University, New York, NY
D. C. Richardson: Department of Biochemistry, Duke University Medical Center, 10027, USA. [13.3]
Durham, NC 27710-3711, USA. [25.2.8] D. E. Tronrud: Howard Hughes Medical Institute, Institute of Molecular Biology,
J. S. Richardson: Department of Biochemistry, Duke University Medical Center, 1229 University of Oregon, Eugene, OR 97403-1229, USA. [25.2.4]
Durham, NC 27710-3711, USA. [25.2.8] H. Tsuruta: SSRL/SLAC & Department of Chemistry, Stanford University, PO
J. Richelle: Unité de Conformation de Macromolécules Biologiques, Université Box 4349, MS69, Stanford, California 94309-0210, USA. [19.3]
Libre de Bruxelles, avenue F. D. Roosevelt 50, CP160/16, B-1050 Bruxelles, M. Tung: Center for Advanced Research in Biotechnology of the Maryland
Belgium. [21.2] Biotechnology Institute and National Institute of Standards and Technology,
D. Ringe: Rosenstiel Basic Medical Sciences Research Center, Brandeis Univer- 9600 Gudelsky Dr., Rockville, MD 20850, USA. [24.4]
sity, 415 South St, Waltham, MA 02254, USA. [23.4] I. UsoÂn: Institut für Anorganisch Chemie, Universität Göttingen, Tammannstrasse
D. W. Rodgers: Department of Biochemistry, Chandler Medical Center, Univer- 4, D-37077 Göttingen, Germany. [16.1]
sity of Kentucky, 800 Rose Street, Lexington, KY 40536-0298, USA. [10.2] A. A. Vagin: Unité de Conformation de Macromolécules Biologiques, Université
M. G. Rossmann: Department of Biological Sciences, Purdue University, West Libre de Bruxelles, avenue F. D. Roosevelt 50, CP160/16, B-1050 Bruxelles,
Lafayette, IN 47907-1392, USA. [1.1, 1.2, 1.4.2, 11.1, 11.5, 13.4] Belgium. [21.2]
C. Sander: MIT Center for Genome Research, One Kendall Square, Cambridge, M. L. Verdonk: Cambridge Crystallographic Data Centre, 12 Union Road,
MA 02139, USA. [23.1.2] Cambridge CB2 1EZ, England. [22.4]
V. R. Sarma: Davy Faraday Research Laboratory, The Royal Institution, London C. L. M. J. Verlinde: Biomolecular Structure Center, Department of Biological
W1X 4BS, England.‡ [26.1] Structure, Howard Hughes Medical Institute, University of Washington,
B. Schneider: The Nucleic Acid Database Project, Department of Chemistry, Seattle, WA 98195-7742, USA. [1.3]
Rutgers University, 610 Taylor Road, Piscataway, NJ 08854-8077, USA. [24.2] C. A. Vernon.² [26.1]
B. P. Schoenborn: Life Sciences Division M888, University of California, Los K. D. Watenpaugh: Structural, Analytical and Medicinal Chemistry, Pharmacia &
Alamos National Laboratory, Los Alamos, NM 8745, USA. [6.2] Upjohn, Inc., Kalamazoo, MI 49001-0119, USA. [18.1]
K. A. Sharp: E. R. Johnson Research Foundation, Department of Biochemistry and C. M. Weeks: Hauptman–Woodward Medical Research Institute, Inc., 73 High
Biophysics, University of Pennsylvania, Philadelphia, PA 19104-6059, USA. Street, Buffalo, NY 14203-1196, USA. [16.1]
[22.3] H. Weissig: San Diego Supercomputer Center, University of California, San Diego,
G. M. Sheldrick: Lehrstuhl für Strukturchemie, Universität Göttingen, 9500 Gilman Drive, La Jolla, CA 92093-0537, USA. [24.5]
Tammannstrasse 4, D-37077 Göttingen, Germany. [16.1, 25.2.10] E. M. Westbrook: Molecular Biology Consortium, Argonne, Illinois 60439, USA.
I. N. Shindyalov: San Diego Supercomputer Center, University of California, San [5.2]
Diego, 9500 Gilman Drive, La Jolla, CA 92093-0537, USA. [24.5] J. Westbrook: The Nucleic Acid Database Project, Department of Chemistry,
T. Simonson: Laboratoire de Biologie Structurale (CNRS), IGBMC, 1 rue Laurent Rutgers University, 610 Taylor Road, Piscataway, NJ 08854-8077, USA. [24.2,
Fries, 67404 Illkirch (CU de Strasbourg), France. [25.2.3] 24.5]
J. L. Smith: Department of Biological Sciences, Purdue University, West Lafayette, K. S. Wilson: Structural Biology Laboratory, Department of Chemistry, University
IN 47907-1392, USA. [14.2.1] of York, York YO10 5DD, England. [9.1, 18.4. 25.2.5]
M. J. E. Sternberg: Institute of Cancer Research, 44 Lincoln’s Inn Fields, London S. J. Wodak: Unité de Conformation de Macromolécules Biologiques, Université
WC2A 3PX, England. [12.1] Libre de Bruxelles, avenue F. D. Roosevelt 50, CP160/16, B-1050 Bruxelles,
A. M. Stock: Center for Advanced Biotechnology and Medicine, Howard Hughes Belgium, and EMBL–EBI, Wellcome Trust Genome Campus, Hinxton,
Medical Institute and University of Medicine and Dentistry of New Jersey – Cambridge CB10 1SD, England. [21.2]
Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854- K. WuÈthrich: Institut für Molekularbiologie und Biophysik, Eidgenössische
5627, USA. [3.1] Technische Hochschule-Hönggerberg, CH-8093 Zürich, Switzerland. [19.7]
U. Stocker: Laboratory of Physical Chemistry, ETH-Zentrum, 8092 Zürich, T. O. Yeates: UCLA–DOE Laboratory of Structural Biology and Molecular
Switzerland. [20.1] Medicine, Department of Chemistry & Biochemistry and Molecular Biology
G. Stubbs: Department of Molecular Biology, Vanderbilt University, Nashville, Institute, UCLA, Los Angeles, CA 90095-1569, USA. [21.3]
TN 37235, USA. [19.5] C. Zardecki: The Nucleic Acid Database Project, Department of Chemistry,
M. T. Stubbs: Institut für Pharmazeutische Chemie der Philipps-Universität Rutgers University, 610 Taylor Road, Piscataway, NJ 08854-8077, USA. [24.2]
Marburg, Marbacher Weg 6, D-35032 Marburg, Germany. [12.2] K. Y. J. Zhang: Division of Basic Sciences, Fred Hutchinson Cancer Research
J. L. Sussman: Department of Structural Biology, Weizmann Institute of Science, Center, 1100 Fairview Ave N., Seattle, WA 90109, USA. [15.1, 25.2.2]
Rehovot 76100, Israel. [24.1] J.-Y. Zou: Department of Cell and Molecular Biology, Uppsala University,
Biomedical Centre, Box 596, SE-751 24 Uppsala, Sweden. [17.1]
‡ Present address: Department of Biochemistry, State University of New York at
Stonybrook, Stonybrook, NY 11794-5215, USA. † Deceased.
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Contents
PAGE
Preface (M. G. Rossmann and E. Arnold) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. xxiii
PART 1. INTRODUCTION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1
1.1. Overview (E. Arnold and M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1
1.2. Historical background (M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 4

1.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 4
1.2.2. 1912 to the 1950s .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 4
1.2.3. The first investigations of biological macromolecules .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 5
1.2.4. Globular proteins in the 1950s .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 5
1.2.5. The first protein structures (1957 to the 1970s) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 7
1.2.6. Technological developments (1958 to the 1980s) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 8
1.2.7. Meetings .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 9
1.3. Macromolecular crystallography and medicine (W. G. J. Hol and C. L. M. J. Verlinde) .. .. .. .. .. .. .. .. .. .. .. .. 10

1.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 10
1.3.2. Crystallography and medicine .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 10
1.3.3. Crystallography and genetic diseases .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 11
1.3.4. Crystallography and development of novel pharmaceuticals .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 12
1.3.5. Vaccines, immunology and crystallography .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 24
1.3.6. Outlook and dreams .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 25
1.4. Perspectives for the future .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 26

1.4.1. Gazing into the crystal ball (E. Arnold) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 26
1.4.2. Brief comments on Gazing into the crystal ball (M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 27
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 27
PART 2. BASIC CRYSTALLOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 45
2.1. Introduction to basic crystallography (J. Drenth) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 45

2.1.1. Crystals .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 45
2.1.2. Symmetry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 46
2.1.3. Point groups and crystal systems .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 47
2.1.4. Basic diffraction physics .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 52
2.1.5. Reciprocal space and the Ewald sphere .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 57
2.1.6. Mosaicity and integrated reflection intensity .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 58
2.1.7. Calculation of electron density .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 59
2.1.8. Symmetry in the diffraction pattern .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 60
2.1.9. The Patterson function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 61
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 62
PART 3. TECHNIQUES OF MOLECULAR BIOLOGY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 65
3.1. Preparing recombinant proteins for X-ray crystallography (S. H. Hughes and A. M. Stock) .. .. .. .. .. .. .. .. .. .. 65
3.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 65
3.1.2. Overview .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 65
3.1.3. Engineering an expression construct .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 66
3.1.4. Expression systems .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 67
3.1.5. Protein purification .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 75
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3.1.6. Characterization of the purified product .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 77
3.1.7. Reprise .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 78
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 79
PART 4. CRYSTALLIZATION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 81
4.1. General methods (R. GiegeÂ and A. McPherson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 81

4.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 81
4.1.2. Crystallization arrangements and methodologies .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 81
4.1.3. Parameters that affect crystallization of macromolecules .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 86
4.1.4. How to crystallize a new macromolecule .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 88
4.1.5. Techniques for physical characterization of crystallization .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 89
4.1.6. Use of microgravity .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 91
4.2. Crystallization of membrane proteins (H. Michel) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 94

4.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 94
4.2.2. Principles of membrane-protein crystallization .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 94
4.2.3. General properties of detergents relevant to membrane-protein crystallization .. .. .. .. .. .. .. .. .. .. .. .. 94
4.2.4. The ‘small amphiphile concept’ .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 98
4.2.5. Membrane-protein crystallization with the help of antibody Fv fragments .. .. .. .. .. .. .. .. .. .. .. .. .. .. 98
4.2.6. Membrane-protein crystallization using cubic bicontinuous lipidic phases .. .. .. .. .. .. .. .. .. .. .. .. .. .. 99
4.2.7. General recommendations .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 99
4.3. Application of protein engineering to improve crystal properties (D. R. Davies and A. Burgess Hickman) .. .. .. .. .. .. 100
4.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 100
4.3.2. Improving solubility .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 100
4.3.3. Use of fusion proteins .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 101
4.3.4. Mutations to accelerate crystallization .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 101
4.3.5. Mutations to improve diffraction quality .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 101
4.3.6. Avoiding protein heterogeneity .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 102
4.3.7. Engineering crystal contacts to enhance crystallization in a particular crystal form .. .. .. .. .. .. .. .. .. .. .. 102
4.3.8. Engineering heavy-atom sites .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 103
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 104
PART 5. CRYSTAL PROPERTIES AND HANDLING .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 111
5.1. Crystal morphology, optical properties of crystals and crystal mounting (H. L. Carrell and J. P. Glusker) .. .. .. .. .. 111
5.1.1. Crystal morphology and optical properties .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 111
5.1.2. Crystal mounting .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 114
5.2. Crystal-density measurements (E. M. Westbrook) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 117

5.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 117
5.2.2. Solvent in macromolecular crystals .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 117
5.2.3. Matthews number .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 117
5.2.4. Algebraic concepts .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 117
5.2.5. Experimental estimation of hydration .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 118
5.2.6. Methods for measuring crystal density .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 118
5.2.7. How to handle the solvent density .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 121
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 121
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PART 6. RADIATION SOURCES AND OPTICS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 125
6.1. X-ray sources (U. W. Arndt) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 125

6.1.1. Overview .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 125
6.1.2. Generation of X-rays .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 125
6.1.3. Properties of the X-ray beam .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 127
6.1.4. Beam conditioning .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 129
6.2. Neutron sources (B. P. Schoenborn and R. Knott) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 133

6.2.1. Reactors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 133
6.2.2. Spallation neutron sources .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 137
6.2.3. Summary .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 139
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 140
PART 7. X-RAY DETECTORS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 143
7.1. Comparison of X-ray detectors (S. M. Gruner, E. F. Eikenberry and M. W. Tate) .. .. .. .. .. .. .. .. .. .. .. .. .. .. 143
7.1.1. Commonly used detectors: general considerations .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 143
7.1.2. Evaluating and comparing detectors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 144
7.1.3. Characteristics of different detector approaches .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 145
7.1.4. Future detectors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 147
7.2. CCD detectors (M. W. Tate, E. F. Eikenberry and S. M. Gruner) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 148

7.2.1. Overview .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 148
7.2.2. CCD detector assembly .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 148
7.2.3. Calibration and correction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 149
7.2.4. Detector system integration .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 151
7.2.5. Applications to macromolecular crystallography .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 152
7.2.6. Future of CCD detectors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 152
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 152
PART 8. SYNCHROTRON CRYSTALLOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 155
8.1. Synchrotron-radiation instrumentation, methods and scientific utilization (J. R. Helliwell) .. .. .. .. .. .. .. .. .. .. 155
8.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 155
8.1.2. The physics of SR .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 155
8.1.3. Insertion devices (IDs) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 155
8.1.4. Beam characteristics delivered at the crystal sample .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 156
8.1.5. Evolution of SR machines and experiments .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 158
8.1.6. SR instrumentation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 161
8.1.7. SR monochromatic and Laue diffraction geometry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 162
8.1.8. Scientific utilization of SR in protein crystallography .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 164
8.2. Laue crystallography: time-resolved studies (K. Moffat) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 167

8.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 167
8.2.2. Principles of Laue diffraction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 167
8.2.3. Practical considerations in the Laue technique .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 168
8.2.4. The time-resolved experiment .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 170
8.2.5. Conclusions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 171
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 172
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PART 9. MONOCHROMATIC DATA COLLECTION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177
9.1. Principles of monochromatic data collection (Z. Dauter and K. S. Wilson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177
9.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177
9.1.2. The components of a monochromatic X-ray experiment .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177
9.1.3. Data completeness .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177
9.1.4. X-ray sources .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177
9.1.5. Goniostat geometry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 178
9.1.6. Basis of the rotation method .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 179
9.1.7. Rotation method: geometrical completeness .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 183
9.1.8. Crystal-to-detector distance .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 188
9.1.9. Wavelength .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 188
9.1.10. Lysozyme as an example .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 189
9.1.11. Rotation method: qualitative factors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 190
9.1.12. Radiation damage .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 191
9.1.13. Relating data collection to the problem in hand .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 192
9.1.14. The importance of low-resolution data .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 194
9.1.15. Data quality over the whole resolution range .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 194
9.1.16. Final remarks .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 194
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 195
PART 10. CRYOCRYSTALLOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 197
10.1. Introduction to cryocrystallography (H. Hope) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 197

10.1.1. Utility of low-temperature data collection .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 197
10.1.2. Cooling of biocrystals .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 197
10.1.3. Principles of cooling equipment .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 199
10.1.4. Operational considerations .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 199
10.1.5. Concluding note .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 201
10.2. Cryocrystallography techniques and devices (D. W. Rodgers) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 202

10.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 202
10.2.2. Crystal preparation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 202
10.2.3. Crystal mounting .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 203
10.2.4. Flash cooling .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 205
10.2.5. Transfer and storage .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 206
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 207
PART 11. DATA PROCESSING .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 209
11.1. Automatic indexing of oscillation images (M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 209

11.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 209
11.1.2. The crystal orientation matrix .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 209
11.1.3. Fourier analysis of the reciprocal-lattice vector distribution when projected onto a chosen direction .. .. .. .. .. 209
11.1.4. Exploring all possible directions to find a good set of basis vectors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 210
11.1.5. The program .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 211
11.2. Integration of macromolecular diffraction data (A. G. W. Leslie) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 212

11.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 212
11.2.2. Prerequisites for accurate integration .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 212
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11.2.3. Methods of integration .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 212
11.2.4. The measurement box .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 212
11.2.5. Integration by simple summation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 213
11.2.6. Integration by profile fitting .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 214
11.3. Integration, scaling, space-group assignment and post refinement (W. Kabsch) .. .. .. .. .. .. .. .. .. .. .. .. .. .. 218
11.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 218
11.3.2. Modelling rotation images .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 218
11.3.3. Integration .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 221
11.3.4. Scaling .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 222
11.3.5. Post refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 223
11.3.6. Space-group assignment .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 224
11.4. DENZO and SCALEPACK (Z. Otwinowski and W. Minor) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 226

11.4.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 226
11.4.2. Diffraction from a perfect crystal lattice .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 226
11.4.3. Autoindexing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 227
11.4.4. Coordinate systems .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 228
11.4.5. Experimental assumptions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 229
11.4.6. Prediction of the diffraction pattern .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 231
11.4.7. Detector diagnostics .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 233
11.4.8. Multiplicative corrections (scaling) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 233
11.4.9. Global refinement or post refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 233
11.4.10. Graphical command centre .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 233
11.4.11. Final note .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 235
11.5. The use of partially recorded reflections for post refinement, scaling and averaging X-ray diffraction data (C. G. van Beek,
R. Bolotovsky and M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 236
11.5.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 236
11.5.2. Generalization of the Hamilton, Rollett and Sparks equations to take into account partial reflections .. .. .. .. .. 236
11.5.3. Selection of reflections useful for scaling .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 237
11.5.4. Restraints and constraints .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 237
11.5.5. Generalization of the procedure for averaging reflection intensities .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 238
11.5.6. Estimating the quality of data scaling and averaging .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 238
11.5.7. Experimental results .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 238
11.5.8. Conclusions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 241
Appendix 11.5.1. Partiality model (Rossmann, 1979; Rossmann et al., 1979) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 241
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 243
PART 12. ISOMORPHOUS REPLACEMENT .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 247
12.1. The preparation of heavy-atom derivatives of protein crystals for use in multiple isomorphous replacement and anomalous
scattering (D. Carvin, S. A. Islam, M. J. E. Sternberg and T. L. Blundell) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 247
12.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 247
12.1.2. Heavy-atom data bank .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 247
12.1.3. Properties of heavy-atom compounds and their complexes .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 248
12.1.4. Amino acids as ligands .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 250
12.1.5. Protein chemistry of heavy-atom reagents .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 250
12.1.6. Metal-ion replacement in metalloproteins .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 254
12.1.7. Analogues of amino acids .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 255
12.1.8. Use of the heavy-atom data bank to select derivatives .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 255
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12.2. Locating heavy-atom sites (M. T. Stubbs and R. Huber) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 256

12.2.1. The origin of the phase problem .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 256
12.2.2. The Patterson function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 257
12.2.3. The difference Fourier .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 258
12.2.4. Reality .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 258
12.2.5. Special complications .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 259
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 260
PART 13. MOLECULAR REPLACEMENT .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263
13.1. Noncrystallographic symmetry (D. M. Blow) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263

13.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263
13.1.2. Definition of noncrystallographic symmetry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263
13.1.3. Use of the Patterson function to interpret noncrystallographic symmetry .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263
13.1.4. Interpretation of generalized noncrystallographic symmetry where the molecular structure is partially known .. 265
13.1.5. The power of noncrystallographic symmetry in structure analysis .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 266
13.2. Rotation functions (J. Navaza) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 269

13.2.1. Overview .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 269
13.2.2. Rotations in three-dimensional Euclidean space .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 269
13.2.3. The rotation function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 270
13.2.4. The locked rotation function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 272
13.2.5. Other rotation functions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 273
13.2.6. Concluding remarks .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 273
Appendix 13.2.1. Formulae for the derivation and computation of the fast rotation function .. .. .. .. .. .. .. .. .. .. 273
13.3. Translation functions (L. Tong) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 275

13.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 275
13.3.2. R-factor and correlation-coefficient translation functions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 275
13.3.3. Patterson-correlation translation function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 276
13.3.4. Phased translation function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 276
13.3.5. Packing check in translation functions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 277
13.3.6. The unique region of a translation function (the Cheshire group) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 277
13.3.7. Combined molecular replacement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 277
13.3.8. The locked translation function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 277
13.3.9. Miscellaneous translation functions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 278
13.4. Noncrystallographic symmetry averaging of electron density for molecular-replacement phase refinement and
extension (M. G. Rossmann and E. Arnold) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 279
13.4.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 279
13.4.2. Noncrystallographic symmetry (NCS) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 279
13.4.3. Phase determination using NCS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 280
13.4.4. The p- and h-cells .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 281
13.4.5. Combining crystallographic and noncrystallographic symmetry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 282
13.4.6. Determining the molecular envelope .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 283
13.4.7. Finding the averaged density .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 284
13.4.8. Interpolation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 285
13.4.9. Combining different crystal forms .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 285
13.4.10. Phase extension and refinement of the NCS parameters .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 285
13.4.11. Convergence .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 286
13.4.12. Ab initio phasing starts .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 286
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13.4.13. Recent salient examples in low-symmetry cases: multidomain averaging and systematic applications of multiple-
crystal-form averaging .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 287
13.4.14. Programs .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 288
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 288
PART 14. ANOMALOUS DISPERSION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 293
14.1. Heavy-atom location and phase determination with single-wavelength diffraction data (B. W. Matthews) .. .. .. .. .. .. 293
14.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 293
14.1.2. The isomorphous-replacement method .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 293
14.1.3. The method of multiple isomorphous replacement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 294
14.1.4. The method of Blow & Crick .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 294
14.1.5. The best Fourier .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 295
14.1.6. Anomalous scattering .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 295
14.1.7. Theory of anomalous scattering .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 295
14.1.8. The phase probability distribution for anomalous scattering .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 296
14.1.9. Anomalous scattering without isomorphous replacement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 297
14.1.10. Location of heavy-atom sites .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 297
14.1.11. Use of anomalous-scattering data in heavy-atom location .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 297
14.1.12. Use of difference Fourier syntheses .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 297
14.1.13. Single isomorphous replacement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 297
14.2. MAD and MIR .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 299

14.2.1. Multiwavelength anomalous diffraction (J. L. Smith and W. A. Hendrickson) .. .. .. .. .. .. .. .. .. .. .. .. 299
14.2.2. Automated MAD and MIR structure solution (T. C. Terwilliger and J. Berendzen) .. .. .. .. .. .. .. .. .. .. 303
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 307
PART 15. DENSITY MODIFICATION AND PHASE COMBINATION .. .. .. .. .. .. .. .. .. .. .. .. .. 311
15.1. Phase improvement by iterative density modification (K. Y. J. Zhang, K. D. Cowtan and P. Main) .. .. .. .. .. .. .. .. 311
15.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 311
15.1.2. Density-modification methods .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 311
15.1.3. Reciprocal-space interpretation of density modification .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 319
15.1.4. Phase combination .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 319
15.1.5. Combining constraints for phase improvement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 321
15.1.6. Example .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 323
15.2. Model phases: probabilities, bias and maps (R. J. Read) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 325
15.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 325
15.2.2. Model bias: importance of phase .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 325
15.2.3. Structure-factor probability relationships .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 325
15.2.4. Figure-of-merit weighting for model phases .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 327
15.2.5. Map coefficients to reduce model bias .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 327
15.2.6. Estimation of overall coordinate error .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 328
15.2.7. Difference-map coefficients .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 328
15.2.8. Refinement bias .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 328
15.2.9. Maximum-likelihood structure refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 329
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 329
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PART 16. DIRECT METHODS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 333
16.1. Ab initio phasing (G. M. Sheldrick, H. A. Hauptman, C. M. Weeks, R. Miller and I. UsoÂn) .. .. .. .. .. .. .. .. .. .. 333
16.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 333
16.1.2. Normalized structure-factor magnitudes .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 333
16.1.3. Starting the phasing process .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 334
16.1.4. Reciprocal-space phase refinement or expansion (shaking) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 335
16.1.5. Real-space constraints (baking) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 336
16.1.6. Fourier refinement (twice baking) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 336
16.1.7. Computer programs for dual-space phasing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 337
16.1.8. Applying dual-space programs successfully .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 339
16.1.9. Extending the power of direct methods .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 344
16.2. The maximum-entropy method (G. Bricogne) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 346

16.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 346
16.2.2. The maximum-entropy principle in a general context .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 346
16.2.3. Adaptation to crystallography .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 348
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 349
PART 17. MODEL BUILDING AND COMPUTER GRAPHICS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 353
17.1. Around O (G. J. Kleywegt, J.-Y. Zou, M. Kjeldgaard and T. A. Jones) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 353
17.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 353
17.1.2. O .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 353
17.1.3. RAVE .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 354
17.1.4. Structure analysis .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 355
17.1.5. Utilities .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 355
17.1.6. Other services .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 356
17.2. Molecular graphics and animation (A. J. Olson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 357

17.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 357
17.2.2. Background – the evolution of molecular graphics hardware and software .. .. .. .. .. .. .. .. .. .. .. .. .. 357
17.2.3. Representation and visualization of molecular data and models .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 358
17.2.4. Presentation graphics .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 363
17.2.5. Looking ahead .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 365
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 366
PART 18. REFINEMENT .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 369
18.1. Introduction to refinement (L. F. Ten Eyck and K. D. Watenpaugh) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 369

18.1.1. Overview .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 369
18.1.2. Background .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 369
18.1.3. Objectives .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 369
18.1.4. Least squares and maximum likelihood .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 369
18.1.5. Optimization .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 370
18.1.6. Data .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 370
18.1.7. Models .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 370
18.1.8. Optimization methods .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 372
18.1.9. Evaluation of the model .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 373
18.1.10. Conclusion .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 374
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18.2. Enhanced macromolecular refinement by simulated annealing (A. T. Brunger, P. D. Adams and L. M. Rice) .. .. .. .. .. 375
18.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 375
18.2.2. Cross validation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 375
18.2.3. The target function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 375
18.2.4. Searching conformational space .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 377
18.2.5. Examples .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 379
18.2.6. Multi-start refinement and structure-factor averaging .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 380
18.2.7. Ensemble models .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 380
18.2.8. Conclusions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 381
18.3. Structure quality and target parameters (R. A. Engh and R. Huber) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 382
18.3.1. Purpose of restraints .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 382
18.3.2. Formulation of refinement restraints .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 382
18.3.3. Strategy of application during building/refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 392
18.3.4. Future perspectives .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 392
18.4. Refinement at atomic resolution (Z. Dauter, G. N. Murshudov and K. S. Wilson) .. .. .. .. .. .. .. .. .. .. .. .. .. 393
18.4.1. Definition of atomic resolution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 393
18.4.2. Data .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 395
18.4.3. Computational algorithms and strategies .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 396
18.4.4. Computational options and tactics .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 396
18.4.5. Features in the refined model .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 398
18.4.6. Quality assessment of the model .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 401
18.4.7. Relation to biological chemistry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 401
18.5. Coordinate uncertainty (D. W. J. Cruickshank) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 403

18.5.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 403
18.5.2. The least-squares method .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 404
18.5.3. Restrained refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 405
18.5.4. Two examples of full-matrix inversion .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 406
18.5.5. Approximate methods .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 409
18.5.6. The diffraction-component precision index .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 410
18.5.7. Examples of the diffraction-component precision index .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 411
18.5.8. Luzzati plots .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 412
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 414
PART 19. OTHER EXPERIMENTAL TECHNIQUES .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 419
19.1. Neutron crystallography: methods and information content (A. A. Kossiakoff) .. .. .. .. .. .. .. .. .. .. .. .. .. .. 419
19.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 419
19.1.2. Diffraction geometries .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 419
19.1.3. Neutron density maps – information content .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 419
19.1.4. Phasing models and evaluation of correctness .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 420
19.1.5. Evaluation of correctness .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 420
19.1.6. Refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 421
19.1.7. D2O H2O solvent difference maps .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 421
19.1.8. Applications of D2O H2O solvent difference maps .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 422
19.2. Electron diffraction of protein crystals (W. Chiu) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 423

19.2.1. Electron scattering .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 423
19.2.2. The electron microscope .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 423
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19.2.3. Data collection .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 423
19.2.4. Data processing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 425
19.2.5. Future development .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 427
19.3. Small-angle X-ray scattering (H. Tsuruta and J. E. Johnson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 428

19.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 428
19.3.2. Small-angle single-crystal X-ray diffraction studies .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 428
19.3.3. Solution X-ray scattering studies .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 429
19.4. Small-angle neutron scattering (D. M. Engelman and P. B. Moore) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 438

19.4.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 438
19.4.2. Fundamental relationships .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 438
19.4.3. Contrast variation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 439
19.4.4. Distance measurements .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 442
19.4.5. Practical considerations .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 442
19.4.6. Examples .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 443
19.5. Fibre diffraction (R. Chandrasekaran and G. Stubbs) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 444

19.5.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 444
19.5.2. Types of fibres .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 444
19.5.3. Diffraction by helical molecules .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 445
19.5.4. Fibre preparation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 446
19.5.5. Data collection .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 446
19.5.6. Data processing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 446
19.5.7. Determination of structures .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 447
19.5.8. Structures determined by X-ray fibre diffraction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 449
19.6. Electron cryomicroscopy (T. S. Baker and R. Henderson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 451

19.6.1. Abbreviations used .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 451
19.6.2. The role of electron microscopy in macromolecular structure determination .. .. .. .. .. .. .. .. .. .. .. .. 451
19.6.3. Electron scattering and radiation damage .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 452
19.6.4. Three-dimensional electron cryomicroscopy of macromolecules .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 453
19.6.5. Recent trends .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 463
19.7. Nuclear magnetic resonance (NMR) spectroscopy (K. WuÈthrich) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 464

19.7.1. Complementary roles of NMR in solution and X-ray crystallography in structural biology .. .. .. .. .. .. .. .. 464
19.7.2. A standard protocol for NMR structure determination of proteins and nucleic acids .. .. .. .. .. .. .. .. .. .. 464
19.7.3. Combined use of single-crystal X-ray diffraction and solution NMR for structure determination .. .. .. .. .. .. 466
19.7.4. NMR studies of solvation in solution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 466
19.7.5. NMR studies of rate processes and conformational equilibria in three-dimensional macromolecular structures .. 466
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 467
PART 20. ENERGY CALCULATIONS AND MOLECULAR DYNAMICS .. .. .. .. .. .. .. .. .. .. .. .. 481
20.1. Molecular-dynamics simulation of protein crystals: convergence of molecular properties of ubiquitin (U. Stocker and
W. F. van Gunsteren) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 481
20.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 481
20.1.2. Methods .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 481
20.1.3. Results .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 482
20.1.4. Conclusions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 488
20.2. Molecular-dynamics simulations of biological macromolecules (C. B. Post and V. M. Dadarlat) .. .. .. .. .. .. .. .. .. 489
20.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 489
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20.2.2. The simulation method .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 489
20.2.3. Potential-energy function .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 489
20.2.4. Empirical parameterization of the force field .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 491
20.2.5. Modifications in the force field for structure determination .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 491
20.2.6. Internal dynamics and average structures .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 491
20.2.7. Assessment of the simulation procedure .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 492
20.2.8. Effect of crystallographic atomic resolution on structural stability during molecular dynamics .. .. .. .. .. .. .. 492
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 494
PART 21. STRUCTURE VALIDATION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 497
21.1. Validation of protein crystal structures (G. J. Kleywegt) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 497

21.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 497
21.1.2. Types of error .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 497
21.1.3. Detecting outliers .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 498
21.1.4. Fixing errors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 499
21.1.5. Preventing errors .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 499
21.1.6. Final model .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 500
21.1.7. A compendium of quality criteria .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 500
21.1.8. Future .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 506
21.2. Assessing the quality of macromolecular structures (S. J. Wodak, A. A. Vagin, J. Richelle, U. Das, J. Pontius and
H. M. Berman) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 507
21.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 507
21.2.2. Validating the geometric and stereochemical parameters of the model .. .. .. .. .. .. .. .. .. .. .. .. .. .. 507
21.2.3. Validation of a model versus experimental data .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 509
21.2.4. Atomic resolution structures .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 517
21.2.5. Concluding remarks .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 518
21.3. Detection of errors in protein models (O. Dym, D. Eisenberg and T. O. Yeates) .. .. .. .. .. .. .. .. .. .. .. .. .. .. 520
21.3.1. Motivation and introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 520
21.3.2. Separating evaluation from refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 520
21.3.3. Algorithms for the detection of errors in protein models and the types of errors they detect .. .. .. .. .. .. .. .. 520
21.3.4. Selection of database .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 521
21.3.5. Examples: detection of errors in structures .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 521
21.3.6. Summary .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 525
21.3.7. Availability of software .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 525
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 526
PART 22. MOLECULAR GEOMETRY AND FEATURES .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 531
22.1. Protein surfaces and volumes: measurement and use .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 531

22.1.1. Protein geometry: volumes, areas and distances (M. Gerstein and F. M. Richards) .. .. .. .. .. .. .. .. .. .. 531
22.1.2. Molecular surfaces: calculations, uses and representations (M. S. Chapman and M. L. Connolly) .. .. .. .. .. .. 539
22.2. Hydrogen bonding in biological macromolecules (E. N. Baker) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 546

22.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 546
22.2.2. Nature of the hydrogen bond .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 546
22.2.3. Hydrogen-bonding groups .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 546
22.2.4. Identification of hydrogen bonds: geometrical considerations .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 547
22.2.5. Hydrogen bonding in proteins .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 547
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22.2.6. Hydrogen bonding in nucleic acids .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 551
22.2.7. Non-conventional hydrogen bonds .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 551
22.3. Electrostatic interactions in proteins (K. A. Sharp) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 553

22.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 553
22.3.2. Theory .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 553
22.3.3. Applications .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 555
22.4. The relevance of the Cambridge Structural Database in protein crystallography (F. H. Allen, J. C. Cole and
M. L. Verdonk) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 558
22.4.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 558
22.4.2. The CSD and the PDB: data acquisition and data quality .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 558
22.4.3. Structural knowledge from the CSD .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 559
22.4.4. Intramolecular geometry .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 560
22.4.5. Intermolecular data .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 562
22.4.6. Conclusion .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 567
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 567
PART 23. STRUCTURAL ANALYSIS AND CLASSIFICATION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 575
23.1. Protein folds and motifs: representation, comparison and classification .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 575
23.1.1. Protein-fold classification (C. Orengo and J. Thornton) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 575
23.1.2. Locating domains in 3D structures (L. Holm and C. Sander) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 577
23.2. Protein–ligand interactions (A. E. Hodel and F. A. Quiocho) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 579

23.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 579
23.2.2. Protein–carbohydrate interactions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 579
23.2.3. Metals .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 580
23.2.4. Protein–nucleic acid interactions .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 581
23.2.5. Phosphate and sulfate .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 585
23.3. Nucleic acids (R. E. Dickerson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 588

23.3.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 588
23.3.2. Helix parameters .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 588
23.3.3. Comparison of A, B and Z helices .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 596
23.3.4. Sequence–structure relationships in B-DNA .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 602
23.3.5. Summary .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 609
Appendix 23.3.1. X-ray analyses of A, B and Z helices .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 609
23.4. Solvent structure (C. Mattos and D. Ringe) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 623

23.4.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 623
23.4.2. Determination of water molecules .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 624
23.4.3. Structural features of protein–water interactions derived from database analysis .. .. .. .. .. .. .. .. .. .. .. 625
23.4.4. Water structure in groups of well studied proteins .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 630
23.4.5. The classic models: small proteins with high-resolution crystal structures .. .. .. .. .. .. .. .. .. .. .. .. .. 637
23.4.6. Water molecules as mediators of complex formation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 638
23.4.7. Conclusions and future perspectives .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 640
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 641
PART 24. CRYSTALLOGRAPHIC DATABASES .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 649
24.1. The Protein Data Bank at Brookhaven (J. L. Sussman, D. Lin, J. Jiang, N. O. Manning, J. Prilusky and E. E. Abola) .. .. 649
24.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 649
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24.1.2. Background and significance of the resource .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 649
24.1.3. The PDB in 1999 .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 650
24.1.4. Examples of the impact of the PDB .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 654
24.2. The Nucleic Acid Database (NDB) (H. M. Berman, Z. Feng, B. Schneider, J. Westbrook and C. Zardecki) .. .. .. .. .. .. 657
24.2.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 657
24.2.2. Information content of the NDB .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 657
24.2.3. Data processing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 657
24.2.4. The database .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 659
24.2.5. Data distribution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 659
24.2.6. Outreach .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 662
24.3. The Cambridge Structural Database (CSD) (F. H. Allen and V. J. Hoy) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 663
24.3.1. Introduction and historical perspective .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 663
24.3.2. Information content of the CSD .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 663
24.3.3. The CSD software system .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 665
24.3.4. Knowledge engineering from the CSD .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 667
24.3.5. Accessing the CSD system and IsoStar .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 668
24.3.6. Conclusion .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 668
24.4. The Biological Macromolecule Crystallization Database (G. L. Gilliland, M. Tung and J. E. Ladner) .. .. .. .. .. .. .. 669
24.4.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 669
24.4.2. History of the BMCD .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 669
24.4.3. BMCD data .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 669
24.4.4. BMCD implementation – web interface .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 670
24.4.5. Reproducing published crystallization procedures .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 670
24.4.6. Crystallization screens .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 671
24.4.7. A general crystallization procedure .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 671
24.4.8. The future of the BMCD .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 674
24.5. The Protein Data Bank, 1999– (H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig,
I. N. Shindyalov and P. E. Bourne) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 675
24.5.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 675
24.5.2. Data acquisition and processing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 675
24.5.3. The PDB database resource .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 677
24.5.4. Data distribution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 679
24.5.5. Data archiving .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 679
24.5.6. Maintenance of the legacy of the BNL system .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 680
24.5.7. Current developments .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 680
24.5.8. PDB advisory boards .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 680
24.5.9. Further information .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 680
24.5.10. Conclusion .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 681
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 681
PART 25. MACROMOLECULAR CRYSTALLOGRAPHY PROGRAMS .. .. .. .. .. .. .. .. .. .. .. .. 685
25.1. Survey of programs for crystal structure determination and analysis of macromolecules (J. Ding and E. Arnold) .. .. .. 685
25.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 685
25.1.2. Multipurpose crystallographic program systems .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 685
25.1.3. Data collection and processing .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 687
25.1.4. Phase determination and structure solution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 688
25.1.5. Structure refinement .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 689
xxi
CONTENTS
25.1.6. Phase improvement and density-map modification .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 689
25.1.7. Graphics and model building .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 690
25.1.8. Structure analysis and verification .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 691
25.1.9. Structure presentation .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 693
25.2. Programs and program systems in wide use .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 695

25.2.1. PHASES (W. Furey) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 695
25.2.2. DM/DMMULTI software for phase improvement by density modification (K. D. Cowtan, K. Y. J. Zhang and
P. Main) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 705
25.2.3. The structure-determination language of the Crystallography & NMR System (A. T. Brunger, P. D. Adams,
W. L. DeLano, P. Gros, R. W. Grosse-Kunstleve, J.-S. Jiang, N. S. Pannu, R. J. Read, L. M. Rice and T. Simonson) .. 710
25.2.4. The TNT refinement package (D. E. Tronrud and L. F. Ten Eyck) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 716
25.2.5. The ARP/wARP suite for automated construction and refinement of protein models (V. S. Lamzin, A. Perrakis and
K. S. Wilson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 720
25.2.6. PROCHECK: validation of protein-structure coordinates (R. A. Laskowski, M. W. MacArthur and J. M. Thornton) 722
25.2.7. MolScript (P. J. Kraulis) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 725
25.2.8. MAGE, PROBE and kinemages (D. C. Richardson and J. S. Richardson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. 727
25.2.9. XDS (W. Kabsch) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 730
25.2.10. Macromolecular applications of SHELX (G. M. Sheldrick) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 734
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 738
PART 26. A HISTORICAL PERSPECTIVE .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 745
26.1. How the structure of lysozyme was actually determined (C. C. F. Blake, R. H. Fenn, L. N. Johnson, D. F. Koenig,
G. A. Mair, A. C. T. North, J. W. H. Oldham, D. C. Phillips, R. J. Poljak, V. R. Sarma and C. A. Vernon) .. .. .. .. .. .. 745
26.1.1. Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 745
26.1.2. Structure analysis at 6 Å resolution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 745
26.1.3. Analysis of the structure at 2 Å resolution .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 753
26.1.4. Structural studies on the biological function of lysozyme .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 765
References .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 771
Author index .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 775
Subject index .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 793
xxii
Preface
By Michael G. Rossmann and Eddy Arnold
International Tables for Crystallography, Volume F, Crystal- Together we fleshed out an outline that was broader than
lography of Biological Macromolecules, was commissioned by MGR’s original plan, which had focused largely on crystal-
the International Union of Crystallography (IUCr) in recognition lographic theory and technique. We felt that it would be valuable
of the extraordinary contributions that knowledge of macro- to briefly cover related techniques beyond X-ray diffraction, as
molecular structure has made, and will make, to the analysis of well as to give an overview of the current field of structural
biological systems, from enzyme catalysis to the workings of a biology. Although basic crystallography is also presented in the
whole cell. The volume covers all stages of a crystallographic other volumes of International Tables, chapters describing
analysis from the preparation of samples using the techniques of fundamental crystallographic principles and practices have been
molecular biology and biochemistry, to crystallization, diffrac- included in an attempt to make the volume as coherent and self-
tion-data collection, phase determination, structure validation contained as possible. We established an advisory board,
and structure analysis. Although the book is written for developed a list of required chapters and obtained promises of
experienced scientists, it is recognized that the modern structural participation from potential authors. In a departure from the style
biologist is more likely to be a biologist interested in structure of previous volumes of International Tables, which have fewer
than a classical crystallographer interested in biology. Thus, articles and authors, we sought contributions for nearly 100
there are chapters on the fundamentals, history and current articles from an even larger number of contributing authors. The
perspectives of macromolecular crystallography, as well as on members of the advisory board reviewed the proposed outline of
the availability of useful programs and databases including the chapters and authors. We were pleasantly surprised when so many
Protein Data Bank. Each chapter has been written by an experts generously agreed to write articles for this volume, and
internationally recognized expert. delighted that the vast majority fulfilled their promises.
Macromolecular crystallography is undergoing a revolution. Significant events punctuating the process were the 1996 and
Just as crystallography became central to the study of chemistry, 1999 IUCr congresses. At the 1996 IUCr Congress in Seattle, we
macromolecular crystallography has become a core science in convened a meeting with many of the authors. There we described
biology. Macromolecular crystallography has shaped our view of the overall project design and received valuable suggestions. At
biological molecular structure, and is providing a broader that time, we hoped that the volume could be completed by 1999.
understanding of biological ultrastructure and the molecular At the 1999 IUCr Congress in Glasgow, we reviewed the detailed
interactions in living systems. As reflected by the exponential contents of the volume at an open meeting on the volumes of
increase in entries in the Protein Data Bank over the past decade, International Tables under development. By that time, we had
there has been an explosion in the number of macromolecular received most of the articles and typesetting began in late 1999.
structures determined, the majority by X-ray crystallography. The complexities of handling a large number of articles from so
Knowledge of the sequences of entire genomes, from bacteria to many authors led to delays at a number of stages. Ultimately, the
human, has sparked a structural genomics effort that aims to completion date became mid-2001.
determine 10 000 new macromolecular structures in the next We are especially grateful to the staff at the IUCr and at our
decade. Crystallography is expected to yield the largest share of own institutions for their dedicated help in bringing this project to
this new crop of structures. The field of macromolecular fruition. At the IUCr, we thank Nicola Ashcroft for an outstanding
crystallography is still evolving rapidly, and capturing its essence job on overall production of the volume, and for her patient
in a single volume is a challenge. Therefore, the volume correspondence and attention to detail. We also thank Peter
emphasizes durable knowledge, but also contains articles on Strickland, Sue King, Theo Hahn, Uri Shmueli, Mike Dacombe
somewhat more volatile topics. and Ted Baker for their help in coordinating the project. At Purdue
This project had its inception when Ted Baker (at that time University, we thank Cheryl Towell and Sharon Wilder for
President of the IUCr) approached one of us (MGR) about constant assistance, and Fay Chen for editorial suggestions. At the
writing a book on macromolecular crystallography for the IUCr. Center for Advanced Biotechnology and Medicine and Rutgers
Not only were there already some excellent books that covered University, we thank Susan Mazzocchi and Barbara Shaver for
most aspects of the subject, but the breadth of the subject was their help in handling correspondence and galley proofs from the
now so vast that no single person could possibly be an expert in authors.
all relevant topics. After further exchanges of e-mails, MGR We are also especially indebted to the authors for their generous
realized that the officers of the IUCr were tacitly assuming that contributions and for documenting relevant expertise. We also
he would be willing to carry out the advice he had given so thank the advisors and the members of the advisory board for their
freely. He then asked his former post-doc and coauthor of an help. We are saddened to note that Paul Sigler, a member of the
earlier article on molecular replacement in Volume B of advisory board, passed away during the project. Paul was a
International Tables, Eddy Arnold, to help him get out of a tight towering figure who, with his medical background, recognized the
corner. After some serious deliberations of his own, Eddy agreed role structure plays in providing insights into fundamental
to be co-editor. chemical and biological processes.
xxiii
International Tables for Crystallography (2006). Vol. F, Chapter 1.1, pp. 1–3.
1. INTRODUCTION
1.1. Overview
BY E. ARNOLD AND M. G. ROSSMANN
As the first International Tables volume devoted to the crystal- generation and definition of neutron beams; related articles in other
lography of large biological molecules, Volume F is intended to International Tables volumes include those in Volume C, Chapter
complement existing volumes of International Tables for Crystal- 4.4.
lography. A background history of the subject is followed by a Part 7 describes common methods for detecting X-rays, with a
concise introduction to the basic theory of X-ray diffraction and focus on detection devices that are currently most frequently used,
other requirements for the practice of crystallography. Basic including storage phosphor image plate and CCD detectors. This
crystallographic theory is presented in considerably greater depth has been another rapidly developing area, particularly in the past
in other volumes of International Tables. Much of the information two decades. A further article describing X-ray detector theory and
in the latter portion of this volume is more specifically related to practice is International Tables Volume C, Chapter 7.1.
macromolecular structure. This chapter is intended to serve as a Synchrotron-radiation sources have played a prominent role in
basic guide to the contents of this book and to how the information advancing the frontiers of macromolecular structure determination
herein relates to material in the other International Tables volumes. in terms of size, quality and throughput. The extremely high
Chapter 1.2 presents a brief history of the field of macro- intensity, tunable wavelength characteristics and pulsed time
molecular crystallography. This is followed by an article describing structure of synchrotron beams have enabled many novel
many of the connections of crystallography with the field of experiments. Some of the unique characteristics of synchrotron
medicine and providing an exciting look into the future possibilities radiation are being harnessed to help solve the phase problem using
of structure-based design of drugs, vaccines and other agents. anomalous scattering measurements, e.g. in multiwavelength
Chapter 1.4 provides some personal perspectives on the future of anomalous diffraction (MAD) experiments (see Chapter 14.2).
science and crystallography, and is followed by a complementary The quality of synchrotron-radiation facilities for macromolecular
response suggesting how crystallography could play a central role studies has also been increasing rapidly, partly in response to the
in unifying diverse scientific fields in the future. perceived value of the structures being determined. Many
Chapter 2.1 introduces diffraction theory and fundamentals of synchrotron beamlines have been designed to meet the needs of
crystallography, including concepts of real and reciprocal space, macromolecular experiments. Chapter 8.1 surveys many of the roles
unit-cell geometry, and symmetry. It is shown how scattering from that synchrotron radiation plays in modern macromolecular
electron density and atoms leads to the formulation of structure structure determination. Chapter 8.2 summarizes applications of
factors. The phase problem is introduced, as well as the basic theory the age-old Laue crystallography technique, which has seen a
behind some of the more common methods for its solution. All of revival in the study of macromolecular crystal structures using
the existing International Tables volumes are central references for portions of the white spectrum of synchrotron X-radiation. Chapter
basic crystallography. 4.2 of International Tables Volume C is also a useful reference for
Molecular biology has had a major impact in terms of understanding synchrotron radiation.
accelerating progress in structural biology, and remains a rapidly Chapter 9.1 summarizes many aspects of data collection from
developing area. Chapter 3.1 is a primer on modern molecular- single crystals using monochromatic X-ray beams. Common
biology techniques for producing materials for crystallographic camera-geometry and coordinate-system-definition schemes are
studies. Since large amounts of highly purified materials are given. Because most macromolecular data collection is carried out
required, emphasis is placed on approaches for efficiently and using the oscillation (or rotation) method, strategies related to this
economically yielding samples of biological macromolecules technique are emphasized. A variety of articles in Volume C of
suitable for crystallization. This is complemented by Chapter 4.3, International Tables serve as additional references.
which describes molecular-engineering approaches for enhancing The use of cryogenic cooling of macromolecular crystals for data
the likelihood of obtaining high-quality crystals of biological collection (‘cryocrystallography’) has become the most frequently
macromolecules. used method of crystal handling for data collection. Part 10
The basic theory and practice of macromolecular crystallization summarizes the theory and practice of cryocrystallography.
are described in Chapters 4.1 and 4.2. This, too, is a rapidly Among its advantages are enhanced crystal lifetime and improved
evolving area, with continual advances in theory and practice. It is resolution. Most current experiments in cryocrystallography use
remarkable to consider the macromolecules that have been liquid-nitrogen-cooled gas streams, though some attempts have
crystallized. We expect macromolecular engineering to play a been made to use liquid-helium-cooled gas streams. Just a decade
central role in coaxing more macromolecules to form crystals ago, it was still widely believed that many macromolecular crystals
suitable for structure determination in the future. The material in could not be studied successfully using cryocrystallography, or that
Part 4 is complemented by Part 5, which summarizes traditional the practice would be troublesome or would lead to inferior results.
properties of and methods for handling macromolecular crystals, as Now, crystallographers routinely screen for suitable cryoprotective
well as how to measure crystal density. conditions for data collection even in initial experiments, and often
Part 6 provides a brief introduction to the theory and practice of crystal diffraction quality is no longer assessed except using
generating X-rays and neutrons for diffraction experiments. Chapter cryogenic cooling. However, some crystals have resisted attempts
6.1 describes the basic theory of X-ray production from both to cool successfully to cryogenic temperatures. Thus, data
conventional and synchrotron X-ray sources, as well as methods for collection using ambient conditions, or moderate cooling (from
defining the energy spectrum and geometry of X-ray beams. approximately 40 °C to a few degrees below ambient tempera-
Numerous excellent articles in other volumes of International ture), are not likely to become obsolete in the near future.
Tables go into more depth in these areas and the reader is referred in Part 11 describes the processing of X-ray diffraction data from
particular to Volume C, Chapter 4.2. Chapter 6.2 describes the macromolecular crystals. Special associated problems concern
1
Copyright © 2006 International Union of Crystallography

1. INTRODUCTION
dealing with large numbers of observations, large unit cells (hence solvent regions have lower density), histogram matching (normal
crowded reciprocal lattices) and diverse factors related to crystal distributions of density are expected) and skeletonization (owing to
imperfection (large and often anisotropic mosaicity, variability of the long-chain nature of macromolecules such as proteins).
unit-cell dimensions etc.). Various camera geometries have been Electron-density averaging, discussed in Chapter 13.4, can be
used in macromolecular crystallography, including precession, thought of as a density-modification technique as well. Chapter 15.1
Weissenberg, three- and four-circle diffractometry, and oscillation surveys the general problem and practice of density modification,
or rotation. The majority of diffraction data sets are collected now including a discussion of solvent flattening, histogram matching,
via the oscillation method and using a variety of detectors. Among skeletonization and phase combination methodology. Chapter 15.2
the topics covered in Part 11 are autoindexing, integration, space- discusses weighting of Fourier terms for calculation of electron-
group assignment, scaling and post refinement. density maps in a more general sense, especially with respect to the
Part 12 describes the theory and practice of the isomorphous problem of minimizing model bias in phase improvement. Electron-
replacement method, and begins the portion of Volume F that density modification techniques can often be implemented
addresses how the phase problem in macromolecular crystal- efficiently in reciprocal space, too.
lography can be solved. The isomorphous replacement method Part 16 describes the use of direct methods in macromolecular
was the first technique used for solving macromolecular crystal crystallography. Some 30 years ago, direct methods revolutionized
structures, and will continue to play a central role for the the practice of small-molecule crystallography by facilitating
foreseeable future. Chapter 12.1 describes the basic practice of structure solution directly from intensity measurements. As a result,
isomorphous replacement, including the selection of heavy-metal phase determination of most small-molecule crystal structures has
reagents as candidate derivatives and crystal-derivatization proce- become quite routine. In the meantime, many attempts have been
dures. Chapter 12.2 surveys some of the techniques used in made to apply direct methods to solving macromolecular crystal
isomorphous replacement calculations, including the location of structures. Prospects in this area are improving, but success has
heavy-atom sites and use of that information in phasing. Readers been obtained in only a limited number of cases, often with
are also referred to Chapter 2.4 of International Tables Volume B extremely high resolution data measured from small proteins.
for additional information about the isomorphous replacement Chapter 16.1 surveys progress in the application of direct methods
method. to solve macromolecular crystal structures.
Part 13 describes the molecular replacement method and many of The use of computer graphics for building models of macro-
its uses in solving macromolecular crystal structures. This part molecular structures has facilitated the efficiency of macromol-
covers general definitions of noncrystallographic symmetry, the use ecular structure solution and refinement immensely (Part 17). Until
of rotation and translation functions, and phase improvement and just a little more than 20 years ago, all models of macromolecular
extension via noncrystallographic symmetry. The molecular structures were built as physical models, with parts of appropriate
replacement method is very commonly used to solve macro- dimensions scaled up to our size! Computer-graphics representa-
molecular crystal structures where redundant information is present tions of structures have made macromolecular structure models
either in a given crystal lattice or among different crystals. In some more precise, especially when coupled with refinement methods,
cases, phase information is obtained by averaging noncrystallo- and have contributed to the rapid proliferation of new structural
graphically redundant electron density either within a single crystal information. With continual improvement in computer hardware
lattice or among multiple crystal lattices. In other cases, atomic and software for three-dimensional visualization of molecules (the
models from known structures can be used to help phase unknown crystallographer’s version of ‘virtual reality’), continuing rapid
crystal structures containing related structures. Molecular replace- progress and evolution in this area is likely. The availability of
ment phasing is often used in conjunction with other phasing computer graphics has also contributed greatly to the magnificent
methods, including isomorphous replacement and density modifica- illustration of crystal structures, one of the factors that has thrust
tion methods. International Tables Volume B, Chapter 2.3 is also a structural biology into many prominent roles in modern life and
useful reference for molecular replacement techniques. chemical sciences. Chapter 17.2 surveys the field of computer
Anomalous-dispersion measurements have played an increas- visualization and animation of molecular structures, with a valuable
ingly important role in solving the phase problem for macro- historical perspective. Chapter 3.3 of International Tables Volume
molecular crystals. Anomalous dispersion has been long recognized B is a useful reference for basics of computer-graphics visualization
as a source of experimental phase information; for more than three of molecules.
decades, macromolecular crystallographers have been exploiting As in other areas of crystallography, refinement methods are used
anomalous-dispersion measurements from crystals containing to obtain the most complete and precise structural information from
heavy metals, using even conventional X-ray sources. In the past macromolecular crystallographic data. The often limited resolution
two decades, synchrotron sources have permitted optimized and other factors lead to underdetermination of structural
anomalous-scattering experiments, where the X-ray energy is parameters relative to small-molecule crystal structures. In addition
selected to be near an absorption edge of a scattering element. to X-ray intensity observations, macromolecular refinement
Chapter 14.1 summarizes applications of anomalous scattering incorporates observations about the normal stereochemistry of
using single wavelengths for macromolecular crystal structure molecules, thereby improving the data-to-parameter ratio. Whereas
determination. The multiwavelength anomalous diffraction (MAD) incorporation of geometrical restraints and constraints in macro-
technique, in particular, is used to solve the phase problem for a molecular refinement was initially implemented about 30 years ago,
broad array of macromolecular crystal structures. In the MAD it is now generally a publication prerequisite that this methodology
experiment, intensities measured from a crystal at a number of be used in structure refinement. Basic principles of crystallographic
wavelengths permit direct solution of the phase problem, frequently refinement, including least-squares minimization, constrained
yielding easily interpretable electron-density maps. The theory and refinement and restrained refinement, are described in Chapter
practice of the MAD technique are described in Chapter 14.2. 18.1. Simulated-annealing methods, discussed in Chapter 18.2, can
Density modification, discussed in Part 15, encompasses an array accelerate convergence to a refined structure, and are now widely
of techniques used to aid solution of the phase problem via used in refining macromolecular crystal structures. Structure quality
electron-density-map modifications. Recognition of usual density- and target parameters for stereochemical constraints and restraints
distribution patterns in macromolecular crystal structures permits are discussed in Chapter 18.3. High-resolution refinement of
the application of such techniques as solvent flattening (disordered macromolecular structures, including handling of hydrogen-atom
2
1.1. OVERVIEW
positions, is discussed in Chapter 18.4. Estimation of coordinate generalizations relating surface areas buried at macromolecular
error in structure refinement is discussed in Chapter 18.5. interfaces and energies of association have emerged. Chapter 22.2
Part 19 is a collection of short reviews of alternative methods for surveys the occurrence of hydrogen bonds in biological macro-
studying macromolecular structure. Each can provide information molecules. Electrostatic interactions in proteins are described in
complementary to that obtained from single-crystal X-ray diffrac- Chapter 22.3. The Cambridge Structural Database is the most
tion methods. In fact, structural information obtained from nuclear complete compendium of small-molecule structural data; its role in
magnetic resonance (NMR) spectroscopy or cryo-electron micro- assessing macromolecular crystal structures is discussed in Chapter
scopy is now frequently used in initiating crystal structure solution 22.4.
via the molecular replacement method (Part 13). Neutron Part 23 surveys current knowledge of protein and nucleic acid
diffraction, discussed in Chapter 19.1, can be used to obtain high- structures. Proliferation of structural data has created problems for
precision information about hydrogen atoms in macromolecular classification schemes, which have been forced to co-evolve with
structures. Electron diffraction studies of thin crystals are yielding new structural knowledge. Methods of protein structural classifica-
structural information to increasingly high resolution, often for tion are described in Chapter 23.1. Systematic aspects of ligand
problems where obtaining three-dimensional crystals is challenging binding to macromolecules are discussed in Chapter 23.2. A survey
(Chapter 19.2). Small-angle X-ray (Chapter 19.3) and neutron of nucleic acid structure, geometry and classification schemes is
(Chapter 19.4) scattering studies can be used to obtain information presented in Chapter 23.3. Solvent structure in macromolecular
about shape and electron-density contrast even in noncrystalline crystals is reviewed in Chapter 23.4.
materials and are especially informative in cases of large macro- With the proliferation of macromolecular structures, it has been
molecular assemblies (e.g. viruses and ribosomes). Fibre diffraction necessary to have databases as international resources for rapid
(Chapter 19.5) can be used to study the structure of fibrous access to, and archival of, primary structural data. The functioning
biological molecules. Cryo-electron microscopy and high-resolu- of the former Brookhaven Protein Data Bank (PDB), which for
tion electron microscopy have been applied to the study of detailed almost thirty years was the depository for protein crystal (and later
structures of noncrystalline molecules of increasing complexity NMR) structures, is summarized in Chapter 24.1. Chapter 24.5
(Chapter 19.6). The combination of electron microscopy and describes the organization and features of the new PDB, run by the
crystallography is helping to bridge molecular structure and Research Collaboratory for Structural Bioinformatics, which super-
multi-molecular ultrastructure in living cells. NMR spectroscopy seded the Brookhaven PDB in 1999. The PDB permits rapid access
has become a central method in the determination of small and to the rapidly increasing store of macromolecular structural data via
medium-sized protein structures (Chapter 19.7), and yields unique the internet, as well as rapid correlation of structural data with other
descriptions of molecular interactions and motion in solution. key life sciences databases. The Nucleic Acid Database (NDB),
Continuing breakthroughs in NMR technology are expanding containing nucleic acid structures with and without bound ligands
greatly the size range of structures that can be studied by NMR. and proteins, is described in Chapter 24.2. The Cambridge
Energy and molecular-dynamics calculations already play an Structural Database (CSD), which is the central database for
integral role in many approaches for refining macromolecular small-molecule structures, is described in Chapter 24.3. The
structures (Part 20). Simulation methods hold promise for greater Biological Macromolecule Crystallization Database (BMCD), a
understanding of the time course of macromolecular motion than repository for macromolecular crystallization data, is described in
can be obtained through painstaking experimental approaches. Chapter 24.4.
However, experimental structures are still the starting point for Part 25 summarizes computer programs and packages in
simulation methods, and the quality of simulations is judged common use in macromolecular structure determination and
relative to experimental observables. Chapters 20.1 and 20.2 analysis. Owing to constant changes in this area, the information
present complementary surveys of the current field of energy and in this chapter is expected to be more volatile than that in the
molecular-dynamics calculations. remainder of the volume. Chapter 25.1 presents a survey of some of
Structure validation (Part 21) is an important part of macro- the most popular programs, with a brief description and references
molecular crystal structure determination. Owing in part to the low for further information. Specific programs and program systems
data-to-parameter ratio and to problems of model phase bias, it can summarized include PHASES (Section 25.2.1); DM/DMMULTI
be difficult to correct misinterpretations of structure that can occur (Section 25.2.2); the Crystallography & NMR System or CNS
at many stages of structure determination. Chapters 21.1, 21.2 and (Section 25.2.3); the TNT refinement package (Section 25.2.4); ARP
21.3 present approaches to structure validation using a range of and wARP for automated model construction and refinement
reference information about macromolecular structure, in addition (Section 25.2.5); PROCHECK (Section 25.2.6); MolScript (Section
to observed diffraction intensities. Structure-validation methods are 25.2.7); MAGE, PROBE and kinemages (Section 25.2.8); XDS
especially important in cases where unusual or highly unexpected (Section 25.2.9); and SHELX (Section 25.2.10).
features are found in a new structure. Chapter 26.1 provides a detailed history of the structure
Part 22 presents a survey of many methods used in the analysis of determination of lysozyme, the first enzyme crystal structure to
macromolecular structure. Since macromolecular structures tend to be solved. This chapter serves as a guide to the process by which the
be very complicated, it is essential to extract features, descriptions lysozyme structure was solved. Although the specific methods used
and representations that can simplify information in helpful ways. to determine macromolecular structures have changed, the overall
Calculations of molecular surface areas, volumes and solvent- process is similar and the reader should find this account
accessible surface areas are discussed in Chapter 22.1. Useful entertaining as well as instructive.
references
1.2. Historical background

BY M. G. ROSSMANN
1.2.1. Introduction This was a major crystallographic success and perhaps the first time
that a crystallographer had succeeded in solving a structure when
Crystallography ranks with astronomy as one of the oldest sciences.
little chemical information was available.
Crystals, in the form of precious stones and common minerals, have
Another event which had a major impact was the determination
attractive properties on account of their symmetry and their
of the absolute hand of the asymmetric carbon atom of sodium
refractive and reflective properties, which result in the undefinable
tartrate by Bijvoet (Bijvoet, 1949; Bijvoet et al., 1951). By indexing
quality called beauty. Natural philosophers have long pondered the
the X-ray reflections with a right-handed system, he showed that the
unusual properties seen in the discontinuous surface morphologies
breakdown of Friedel’s law in the presence of an anomalous
of crystals. Hooke (1665) and Huygens (1690) came close to
scatterer was consistent with the asymmetric carbon atom having a
grasping the way repeating objects create discrete crystal faces with
hand in agreement with Fischer’s convention. With that knowledge,
reproducible interfacial angles. The symmetry of mineral crystals
together with the prior results of organic reaction analyses, the
was explored systematically in the 18th and 19th centuries by
absolute hand of other asymmetric carbon atoms could be
measuring the angles between crystal faces, leading to the
established. In particular, the absolute structure of naturally
classification into symmetry systems from triclinic to cubic and
occurring amino acids and riboses was now determined.
the construction of symmetry tables (Schoenflies, 1891; Hilton,
Until the mid-1950s, most structure determinations were made
1903; Astbury et al., 1935) – the predecessors of today’s
using only projection data. This not only reduced the tremendous
International Tables.
effort required for manual indexing and for making visual estimates
of intensity measurements, but also reduced the calculation effort to
1.2.2. 1912 to the 1950s
almost manageable proportions in the absence of computing
It was not until the interpretation of the first X-ray diffraction machines. However, the structure determination of penicillin
experiments by Max von Laue and Peter Ewald in 1912 that it was (Crowfoot, 1948; Crowfoot et al., 1949), carried out during
possible to ascertain the size of the repeating unit in simple crystals. World War II by Dorothy Hodgkin and Charles Bunn, employed
Lawrence Bragg, encouraged by his father, William Bragg, recast some three-dimensional data. A further major achievement was the
the Laue equations into the physically intuitive form now known as solution of the three-dimensional structure of vitamin B12 by
‘Bragg’s law’ (Bragg & Bragg, 1913). This set the stage for a large Dorothy Hodgkin and her colleagues (Hodgkin et al., 1957) in the
number of structure determinations of inorganic salts and metals. 1950s. They first used a cobalt atom as a heavy atom on a vitamin
The discovery of simple structures (Bragg, 1913), such as that of B12 fragment and were able to recognize the ‘corrin’ ring structure.
NaCl, led to a good deal of acrimony, for crystals of such salts were The remainder of the B12 structure was determined by an
shown to consist of a uniform distribution of positive and negative extraordinary collaboration between Dorothy Hodgkin in Oxford
ions, rather than discrete molecules. These early structure and Kenneth Trueblood at UCLA in Los Angeles. While Dorothy’s
determinations were based on trial and error (sometimes guided group did the data collection and interpretation, Ken’s group
by the predictions of Pope and Barlow that were based on packing performed the computing on the very early electronic Standard
considerations) until a set of atomic positions could be found that Western Automatic Computer (SWAC). Additional help was made
satisfied the observed intensity distribution of the X-ray reflections. available by the parallel work of J. G. White at Princeton University
This gave rise to rather pessimistic estimates that structures with in New Jersey. This was at a time before the internet, before e-mail,
more than about four independent atomic parameters would not be before usable transatlantic telephones and before jet travel.
solvable. Transatlantic, propeller-driven air connections had started to
The gradual advance in X-ray crystallography required a operate only a few years earlier.
systematic understanding and tabulation of space groups. Pre- Many technical advances were made in the 1930s that
viously, only various aspects of three-dimensional symmetry contributed to the rapidly increasing achievements of crystal-
operations appropriate for periodic lattices had been listed. lography. W. H. Bragg had earlier suggested (Bragg, 1915) the use
Consequently, in 1935, the growing crystallographic community of Fourier methods to analyse the periodic electron-density
put together the first set of Internationale Tabellen (Hermann, distribution in crystals, and this was utilized by his son, W. L.
1935), containing diagrams and information on about 230 space Bragg (Bragg, 1929a,b). The relationship between a Fourier
groups. After World War II, these tables were enlarged and synthesis and a Fourier analysis demonstrated that the central
combined with Kathleen Lonsdale’s structure-factor formulae problem in structural crystallography was in the phase. Computa-
(Lonsdale, 1936) in the form of International Tables Volume I tional devices to help plot this distribution were invented by Arnold
(Henry & Lonsdale, 1952). Most recently, they have again been Beevers and Henry Lipson in the form of their ‘Beevers–Lipson
revised and extended in Volume A (Hahn, 1983). strips’ (Beevers & Lipson, 1934) and by J. Monteath Robertson
Simple organic compounds started to be examined in the 1920s. with his ‘Robertson sorting board’ (Robertson, 1936). These
Perhaps foremost among these is the structure of hexamethylben- devices were later supplemented by the XRAC electronic analogue
zene by Kathleen Lonsdale (Lonsdale, 1928). She showed that, as machine of Ray Pepinsky (Pepinsky, 1947) and mechanical
had been expected, benzene had a planar hexagonal structure. analogue machines (McLachlan & Champaygne, 1946; Lipson &
Another notable achievement of crystallography was made by J. D. Cochran, 1953) until electronic digital computers came into use
Bernal in the early 1930s. He was able to differentiate between a during the mid-1950s.
number of possible structures for steroids by studying their packing A. Lindo Patterson, inspired by his visit to England in the 1930s
arrangements in different unit cells (Bernal, 1933). Bernal (‘Sage’) where he met Lawrence Bragg, Kathleen Lonsdale and J. Monteath
had an enormous impact on English crystallographers in the 1930s. Robertson, showed how to use F 2 Fourier syntheses for structure
His character was immortalized by the novelist C. P. Snow in his determinations (Patterson, 1934, 1935). When the ‘Patterson’
book The Search (Snow, 1934). By the mid-1930s, J. Monteath synthesis was combined with the heavy-atom method, and (later)
Robertson and I. Woodward had determined the structure of nickel with electronic computers, it transformed analytical organic
phthalocyanine (Robertson, 1935) using the heavy-atom method. chemistry. No longer was it necessary for teams of chemists to
4
1.2. HISTORICAL BACKGROUND
labour for decades on the structure determination of natural (Boyes-Watson et al., 1947; Perutz, 1949). Perutz was correct about
products. Instead, a single crystallographer could solve such a the -keratin-like rods, but not about these being parallel.
structure in a period of months. In Pasadena, Pauling (Pauling & Corey, 1951; Pauling et al.,
Improvements in data-collection devices have also had a major 1951) was building helical polypeptide models to explain Astbury’s
impact. Until the mid-1950s, the most common method of patterns and perhaps to understand the helical structures in
measuring intensities was by visual comparison of reflection globular proteins, such as haemoglobin. Pauling, using his knowl-
‘spots’ on films with a standard scale. However, the use of counters edge of the structure of amino acids and peptide bonds, was forced
(used, for instance, by Bragg in 1912) was gradually automated and to the conclusion that there need not be an integral number of
became the preferred technique in the 1960s. In addition, semi- amino-acid residues per helical turn. He therefore suggested that the
automatic methods of measuring the optical densities along ‘ -helix’, with 3.6 residues per turn, would roughly explain
reciprocal lines on precession photographs were used extensively Astbury’s pattern and that his proposed ‘ -sheet’ structure should
for early protein-structure determinations in the 1950s and 1960s. be related to Astbury’s pattern. Perutz saw that an -helical
structure should give rise to a strong 1.5 Å-spacing reflection as a
consequence of the rise per residue in an -helix (Perutz, 1951a,b).
Demonstration of this reflection in horse hair, then in fibres of
polybenzyl-L-glutamate, in muscle (with Hugh Huxley) and finally
1.2.3. The first investigations of biological in haemoglobin crystals showed that Pauling’s proposed -helix
macromolecules really existed in haemoglobin and presumably also in other globular
Leeds, in the county of Yorkshire, was one of the centres of proteins. Confirmation of helix-like structures came with the
England’s textile industry and home to a small research institute observation of cylindrical rods in the 6 Å-resolution structure of
established to investigate the properties of natural fibres. W. T. myoglobin in 1957 (Kendrew et al., 1958) and eventually at atomic
Astbury became a member of this institute after learning about resolution with the 2 Å myoglobin structure in 1959 (Kendrew et
X-ray diffraction from single crystals in Bragg’s laboratory. He al., 1960). The first atomic resolution confirmation of Pauling’s
investigated the diffraction of X-rays by wool, silk, keratin and structure did not come until 1966 with the structure determination
other natural fibrous proteins. He showed that the resultant patterns of hen egg-white lysozyme (Blake, Mair et al., 1967).
could be roughly classified into two classes, and , and that on Although the stimulus for the Cochran et al. (1952) analysis of
stretching some, for example, wool, the pattern is converted from diffraction from helical structures came from Perutz’s studies of
to (Astbury, 1933). helices in polybenzyl-L-glutamate and their presence in haemoglo-
Purification techniques for globular proteins were also being bin, the impact on the structure determination of nucleic acids was
developed in the 1920s and 1930s, permitting J. B. Sumner at even more significant. The events leading to the discovery of the
Cornell University to crystallize the first enzyme, namely urease, in double-helical structure of DNA have been well chronicled
1926. Not much later, in Cambridge, J. D. Bernal and his student, (Watson, 1968; Olby, 1974; Judson, 1979). The resultant science,
Dorothy Crowfoot (Hodgkin), investigated crystals of pepsin. The often known exclusively as molecular biology, has created a whole
resultant 1934 paper in Nature (Bernal & Crowfoot, 1934) is quite new industry. Furthermore, the molecular-modelling techniques
remarkable because of its speed of publication and because of the used by Pauling in predicting the structure of -helices and -sheets
authors’ extraordinary insight. The crystals of pepsin were found to and by Crick and Watson in determining the structure of DNA had a
deteriorate quickly in air when taken out of their crystallization major effect on more traditional crystallography and the structure
solution and, therefore, had to be contained in a sealed capillary determinations of fibrous proteins, nucleic acids and polysacchar-
tube for all X-ray experiments. This form of protein-crystal ides.
mounting remained in vogue until the 1990s when crystal-freezing Another major early result of profound biological significance
techniques were introduced. But, most importantly, it was was the demonstration by Bernal and Fankuchen in the 1930s
recognized that the pepsin diffraction pattern implied that the (Bernal & Fankuchen, 1941) that tobacco mosaic virus (TMV) had
protein molecules have a unique structure and that these crystals a rod-like structure. This was the first occasion where it was
would be a vehicle for the determination of that structure to atomic possible to obtain a definite idea of the architecture of a virus. Many
resolution. This understanding of protein structure occurred at a of the biological properties of TMV had been explored by Wendell
time when proteins were widely thought to form heterogeneous Stanley working at the Rockefeller Institute in New York. He had
micelles, a concept which persisted another 20 years until Sanger also been able to obtain a large amount of purified virus. Although it
was able to determine the unique amino-acid sequences of the two was not possible to crystallize this virus, it was possible to obtain a
chains in an insulin molecule (Sanger & Tuppy, 1951; Sanger & diffraction pattern of the virus in a viscous solution which had been
Thompson, 1953a,b). agitated to cause alignment of the virus particles. This led Jim
Soon after Bernal and Hodgkin photographed an X-ray Watson (Watson, 1954) to a simple helical structure of protein
diffraction pattern of pepsin, Max Perutz started his historic subunits. Eventually, after continuing studies by Aaron Klug,
investigation of haemoglobin.* Such investigations were, however, Rosalind Franklin, Ken Holmes and others, the structure was
thought to be without hope of any success by most of the determined at atomic resolution (Holmes et al., 1975), in which the
contemporary crystallographers, who avoided crystals that did not helical strand of RNA was protected by the helical array of protein
have a short (less than 4.5 Å) axis for projecting resolved atoms. subunits.
Nevertheless, Perutz computed Patterson functions that suggested
haemoglobin contained parallel -keratin-like bundles of rods
1.2.4. Globular proteins in the 1950s

* Perutz writes, ‘I started X-ray work on haemoglobin in October 1937 and Bragg
became Cavendish Professor in October 1938. Bernal was my PhD supervisor in In 1936, Max Perutz had joined Sir Lawrence Bragg in Cambridge.
1937, but he had nothing to do with my choice of haemoglobin. I began this work at Inspired in part by Keilin (Perutz, 1997), Perutz started to study
the suggestion of Haurowitz, the husband of my cousin Gina Perutz, who was then
in Prague. The first paper on X-ray diffraction from haemoglobin (and crystalline haemoglobin. This work was interrupted by World War
chymotrypsin) was Bernal, Fankuchen & Perutz (Bernal et al., 1938). I did the II, but once the war was over Perutz tenaciously developed a series
experimental work, (and) Bernal showed me how to interpret the X-ray pictures. of highly ingenious techniques. All of these procedures have their
5
1. INTRODUCTION
counterparts in modern ‘protein crystallography’, although few
today recognize their real origin.
The first of these methods was the use of ‘shrinkage’ stages
(Perutz, 1946; Bragg & Perutz, 1952). It had been noted by Bernal
and Crowfoot (Hodgkin) in their study of pepsin that crystals of
proteins deteriorate on exposure to air. Perutz examined crystals
of horse haemoglobin after they were air-dried for short periods of
time and then sealed in capillaries. He found that there were at least
seven consecutive discrete shrinkage stages of the unit cell. He
realized that each shrinkage stage permitted the sampling of the
molecular transform at successive positions, thus permitting him to
map the variation of the continuous transform. As he examined only
the centric (h0l) reflections of the monoclinic crystals, he could
observe when the sign changed from 0 to in the centric projection Fig. 1.2.4.1. Change of structure amplitude for horse haemoglobin as a
(Fig. 1.2.4.1). Thus, he was able to determine the phases (signs) of function of salt concentration in the suspension medium of the low-
order h0l reflections at various lattice shrinkage stages (C, C0 , D, E, F, G,
the central part of the (h0l) reciprocal lattice. This technique is H, J). Reprinted with permission from Perutz (1954). Copyright (1954)
essentially identical to the use of diffraction data from different unit Royal Society of London.
cells for averaging electron density in the ‘modern’ molecular
replacement method. In the haemoglobin case, Patterson projec-
tions had shown that the molecules maintained their orientation Cancer Institute in Buffalo, New York. He proposed to solve the
relative to the a axis as the crystals shrank, but in the more general structure of proteins on the assumption that they consisted of ‘globs’
molecular replacement case, it is necessary to determine the relative which he could treat as single atoms; therefore, he could solve the
orientations of the molecules in each cell. structure by using his inequalities (Harker & Kasper, 1947), i.e., by
The second of Perutz’s techniques depended on observing direct methods. He was aware of the need to use three-dimensional
changes in the intensities of low-order reflections when the data, which meant a full phase determination, rather than the sign
concentration of the dissolved salts (e.g. Cs2 SO4 ) in the solution determination of two-dimensional projection data on which Perutz
between the crystallized molecules was altered (Boyes-Watson et had concentrated. Harker therefore decided to develop automatic
al., 1947; Perutz, 1954). The differences in structure amplitude, diffractometers, as opposed to the film methods being used at
taken together with the previously determined signs, could then map Cambridge. In 1956, he published a procedure for plotting the
out the parts of the crystal unit cell occupied by the haemoglobin isomorphous data of each reflection in a simple graphical manner
molecule. In many respects, this procedure has its equivalent in that allowed an easy determination of its phase (Harker, 1956).
‘solvent flattening’ used extensively in ‘modern’ protein crystal- Unfortunately, the error associated with the data tended to create a
lography. lot of uncertainty.
The third of Perutz’s innovations was the isomorphous In the first systematic phase determination of a protein, namely
replacement method (Green et al., 1954). The origin of the that of myoglobin, phase estimates were made for about 400
isomorphous replacement method goes back to the beginnings of reflections. In order to remove subjectivity, independent estimates
X-ray crystallography when Bragg compared the diffracted were made by Kendrew and Bragg by visual inspection of the
intensities from crystals of NaCl and KCl. J. Monteath Robertson Harker diagram for each reflection. These were later compared
explored the procedure a little further in his studies of before computing an electron-density map. This process was put
phthalocyanines. Perutz used a well known fact that dyes could onto a more objective basis by calculating phase probabilities, as
be diffused into protein crystals, and, hence, heavy-atom described by Blow & Crick (1959) and Dickerson et al. (1961).
compounds might also diffuse into and bind to specific residues One problem with the isomorphous replacement method was the
in the protein. Nevertheless, the sceptics questioned whether even determination of accurate parameters that described the heavy-atom
the heaviest atoms could make a measurable difference to the X-ray replacements. Centric projections were a means of directly
diffraction pattern of a protein.* Perutz therefore developed an determining the coordinates, but no satisfactory method was
instrument which quantitatively recorded the blackening caused by available to determine the relative positions of atoms in different
the reflected X-ray beam on a film. He also showed that the effect of derivatives when there were no centric projections. In particular, it
specifically bound atoms could be observed visually on a film was necessary to establish the relative y coordinates for the heavy-
record of a diffraction pattern. In 1953, this resulted in a complete atom sites in the various derivatives of monoclinic myoglobin and
sign determination of the (h0l) horse haemoglobin structure in monoclinic horse haemoglobin. Perutz (1956) and Bragg (1958)
amplitudes (Green et al., 1954). However, not surprisingly, the had each proposed solutions to this problem, but these were not
projection of the molecule was not very interesting, making it entirely satisfactory. Consequently, it was necessary to average the
necessary to extend the procedure to noncentric, three-dimensional results of different methods to determine the 6 Å phases for
data. It took another five years to determine the first globular protein myoglobin. However, this problem was solved satisfactorily in
structure to near atomic resolution. the structure determination of haemoglobin by using an FH1
In 1950, David Harker was awarded one million US dollars to FH2 2 Patterson-like synthesis in which the vectors between atoms
study the structure of proteins. He worked first at the Brooklyn in the two heavy-atom compounds, H1 and H2, produce negative
Polytechnic Institute in New York and later at the Roswell Park peaks (Rossmann, 1960). This technique also gave rise to the first
least-squares refinement procedure to determine the parameters that
define each heavy atom.
Perutz used punched cards to compute the first three-dimensional
* Perutz writes, ‘I measured the absolute intensity of reflexions from haemoglobin Patterson map of haemoglobin. This was a tremendous computa-
which turned out to be weaker than predicted by Wilson’s statistics. This made me tional undertaking. However, the first digital electronic computers
realise that about 99% of the scattering contributions of the light atoms are
extinguished by interference and that, by contrast, the electrons of a heavy atom, started to appear in the early to mid-1950s. The EDSAC1 and
being concentrated at a point, would scatter in phase and therefore make a EDSAC2 machines were built in the Mathematical Laboratory of
measurable difference to the structure amplitudes.’ Cambridge University. EDSAC1 was used by John Kendrew for the
6
Fig. 1.2.5.2. Cylindrical sections through a helical segment of a myoglobin

polypeptide chain. (a) The density in a cylindrical mantle of 1.95 Å
radius, corresponding to the mean radius of the main-chain atoms in an
-helix. The calculated atomic positions of the -helix are super-
Fig. 1.2.5.1. A model of the myoglobin molecule at 6 Å resolution. imposed and roughly correspond to the density peaks. (b) The density at
Reprinted with permission from Bodo et al. (1959). Copyright (1959) the radius of the -carbon atoms; the positions of the -carbon atoms
Royal Society of London. calculated for a right-handed -helix are marked by the superimposed
grid (Kendrew & Watson, unpublished). Reprinted with permission
from Perutz (1962). Copyright (1962) Elsevier Publishing Co.
6 Å-resolution map of myoglobin (Bluhm et al., 1958). EDSAC2
came on-line in 1958 and was the computer on which all the
calculations were made for the 5.5 Å map of haemoglobin (Cullis et -helices on cylindrical sections (Fig. 1.2.5.2), it was possible to see
al., 1962) and the 2.0 Å map of myoglobin. It was the tool on which not only that the Pauling prediction of the -helix structure was
many of the now well established crystallographic techniques were accurately obeyed, but also that the C atoms were consistent with
initially developed. By about 1960, the home-built, one-of-a-kind laevo amino acids and that all eight helices were right-handed on
machines were starting to be replaced by commercial machines. account of the steric hindrance that would occur between the C
Large mainframe IBM computers (704, 709 etc.), together with atom and carbonyl oxygen in left-handed helices.
FORTRAN as a symbolic language, became available. The first enzyme structure to be solved was that of lysozyme in
1965 (Blake et al., 1965), following a gap of six years after the
excitement caused by the discovery of the globin structures.
Diffusion of substrates into crystals of lysozyme showed how
1.2.5. The first protein structures (1957 to the 1970s)
substrates bound to the enzyme (Blake, Johnson et al., 1967), which
By the time three-dimensional structures of proteins were being in turn suggested a catalytic mechanism and identified the essential
solved, Linderström-Lang (Linderström-Lang & Schellman, 1959) catalytic residues.
had introduced the concepts of ‘primary’, ‘secondary’ and ‘tertiary’ From 1965 onwards, the rate of protein-structure determinations
structures, providing a basis for the interpretation of electron- gradually increased to about one a year: carboxypeptidase (Reeke et
density maps. The first three-dimensional protein structure to be al., 1967), chymotrypsin (Matthews et al., 1967), ribonuclease
solved was that of myoglobin at 6 Å resolution (Fig. 1.2.5.1) in 1957 (Kartha et al., 1967; Wyckoff et al., 1967), papain (Drenth et al.,
(Kendrew et al., 1958). It clearly showed sausage-like features 1968), insulin (Adams et al., 1969), lactate dehydrogenase (Adams
which were assumed to be -helices. The iron-containing haem et al., 1970) and cytochrome c (Dickerson et al., 1971) were early
group was identified as a somewhat larger electron-density feature. examples. Every new structure was a major event. These structures
The structure determination of haemoglobin at 5.5 Å resolution in laid the foundation for structural biology. From a crystallographic
1959 (Cullis et al., 1962) showed that each of its two independent point of view, Drenth’s structure determination of papain was
chains, and , had a fold similar to that of myoglobin and, thus, particularly significant in that he was able to show an amino-acid
suggested a divergent evolutionary process for oxygen transport sequencing error where 13 residues had to be inserted between
molecules. These first protein structures were mostly helical, Phe28 and Arg31, and he showed that a 38-residue peptide that had
features that could be recognized readily at low resolution. Had been assigned to position 138 to 176 needed to be transposed to a
the first structures been of mostly structure, as is the case for position between the extra 13 residues and Arg31.
pepsin or chymotrypsin, history might have been different. The structures of the globins had suggested that proteins with
The absolute hand of the haemoglobin structure was determined similar functions were likely to have evolved from a common
using anomalous dispersion (Cullis et al., 1962) in a manner similar precursor and, hence, that there might be a limited number of
to that used by Bijvoet. This was confirmed almost immediately protein folding motifs. Comparison of the active centres of
when a 2 Å-resolution map of myoglobin was calculated in 1959 chymotrypsin and subtilisin showed that convergent evolutionary
(Kendrew et al., 1960). By plotting the electron density of the pathways could exist (Drenth et al., 1972; Kraut et al., 1972).
7
1. INTRODUCTION
The variety of structures that were being studied increased Rossmann & Blow (1962) recognized that an obvious application
rapidly. The first tRNA structures were determined in the 1960s of the technique would be to viruses with their icosahedral
(Kim et al., 1973; Robertus et al., 1974), the first spherical virus symmetry. They pointed out that the symmetry of the biological
structure was published in 1978 (Harrison et al., 1978) and the oligomer can often be, and sometimes must be, ‘noncrystallo-
photoreaction centre membrane protein structure appeared in 1985 graphic’ or ‘local’, as opposed to being true for the whole infinite
(Deisenhofer et al., 1985). The rate of new structure determinations crystal lattice. Although the conservation of folds had become
has continued to increase exponentially. In 1996, about one new apparent in the study of the globins and a little later in the study of
structure was published every day. Partly in anticipation and partly dehydrogenases (Rossmann et al., 1974), in the 1960s the early
to assure the availability of results, the Brookhaven Protein Data development of the molecular replacement technique was aimed
Bank (PDB) was brought to life at the 1971 Cold Spring Harbor primarily at ab initio phase determination (Rossmann & Blow,
Meeting (H. Berman & J. L. Sussman, private communication). 1963; Main & Rossmann, 1966; Crowther, 1969). It was only in the
Initially, it was difficult to persuade authors to submit their 1970s, when more structures became available, that it was possible
coordinates, but gradually this situation changed to one where to use the technique to solve homologous structures with suitable
most journals require coordinate submission to the PDB, resulting search models. Initially, there was a good deal of resistance to the
in today’s access to structural results via the World Wide Web. use of the molecular replacement technique. Results from the
The growth of structural information permitted generalizations, rotation function were often treated with scepticism, the translation
such as that -sheets have a left-handed twist when going from one problem was thought to have no definitive answer, and there were
strand to the next (Chothia, 1973) and that ‘cross-over’ - - turns excellent reasons to consider that phasing was impossible except for
were almost invariably right-handed (Richardson, 1977). These centric reflections (Rossmann, 1972). It took 25 years before the full
observations and the growth of the PDB have opened up a new field power of all aspects of the molecular replacement technique was
of science. Among the many important results that have emerged fully utilized and accepted (Rossmann et al., 1985).
from this wealth of data is a careful measurement of the main-chain The first real success of the rotation function was in finding the
dihedral angles, confirming the predictions of Ramachandran rotational relationship between the two independent insulin
(Ramachandran & Sasisekharan, 1968), and of side-chain rotamers monomers in the P3 unit cell (Dodson et al., 1966). Crowther
(Ponder & Richards, 1987). Furthermore, it is now possible to produced the fast rotation function, which reduced the computa-
determine whether the folds of domains in a new structure relate to tional times to manageable proportions (Crowther, 1972). Crowther
any previous results quite conveniently (Murzin et al., 1995; Holm (1969) and Main & Rossmann (1966) were able to formulate the
& Sander, 1997). problem of phasing in the presence of noncrystallographic
symmetry in terms of a simple set of simultaneous complex
equations. However, real advances came with applying the
conditions of noncrystallographic symmetry in real space, which
1.2.6. Technological developments (1958 to the 1980s)
was the key to the solution of glyceraldehyde-3-phosphate
In the early 1960s, there were very few who had experience in dehydrogenase (Buehner et al., 1974), tobacco mosaic virus disk
solving a protein structure. Thus, almost a decade passed after the protein (Bloomer et al., 1978) and other structures, aided by Gerard
success with the globins before there was a noticeable surge of new Bricogne’s program for electron-density averaging (Bricogne,
structure reports. In the meantime, there were persistent attempts to 1976), which became a standard of excellence.
find alternative methods to determine protein structure. No account of the early history of protein crystallography is
Blow & Rossmann (1961) demonstrated the power of the single complete without a mention of ways of representing electron
isomorphous replacement method. While previously it had been density. The 2 Å map of myoglobin was interpreted by building a
thought that it was necessary to have at least two heavy-atom model (on a scale of 5 cm to 1 Å with parts designed by Corey and
compounds, if not many more, they showed that a good Pauling at the California Institute of Technology) into a forest of
representation of the structure of haemoglobin could have been vertical rods decorated with coloured clips at each grid point,
made by using only one good derivative. There were also early
attempts at exploiting anomalous dispersion for phase determina-
tion. Rossmann (1961) showed that anomalous differences could be
used to calculate a ‘Bijvoet Patterson’ from which the sites of the
anomalous scatterers (and, hence, heavy-atom sites) could be
determined. Blow & Rossmann (1961), North (1965) and Matthews
(1966) used anomalous-dispersion data to help in phase determina-
tion. Hendrickson stimulated further interest by using Cu K
radiation and employing the anomalous effect of sulfur atoms in
cysteines to solve the entire structure of the crambin molecule
(Hendrickson & Teeter, 1981). With today’s availability of
synchrotrons, and hence the ability to tune to absorption edges,
these early attempts to utilize anomalous data have been vastly
extended to the powerful multiple-wavelength anomalous disper-
sion (MAD) method (Hendrickson, 1991). More recently, the
generality of the MAD technique has been greatly expanded by
using proteins in which methionine residues have been replaced by
selenomethionine, thus introducing selenium atoms as anomalous
scatterers. Fig. 1.2.6.1. The 2 Å-resolution map of sperm-whale myoglobin was
Another advance was the introduction of the ‘molecular represented by coloured Meccano-set clips on a forest of vertical rods.
replacement’ technique (Rossmann, 1972). The inspiration for Each clip was at a grid point. The colour of the clip indicated the height
this method arose out of the observation that many larger proteins of the electron density. The density was interpreted in terms of ‘Corey–

(e.g. haemoglobin) are oligomers of identical subunits and that Pauling’ models on a scale of 5 cm 1 A. Pictured is John Kendrew.
many proteins can crystallize in numerous different forms. (This figure was provided by M. F. Perutz.)
8
representing the height of the electron density (Fig. 1.2.6.1). Later occurred two years later, but this time the number of participants
structures, such as those of lysozyme and carboxypeptidase, were had doubled. By 1970, the meeting had to be transferred to the
built with ‘Kendrew’ models (2 cm to 1 Å) based on electron- village of Alpbach, which had more accommodation; however,
density maps displayed as stacks of large Plexiglas sheets. A major most of the participants still knew each other.
advance came with Fred Richards’ invention of the optical Another set of international meetings (or schools, as the Italians
comparator (a ‘Richards box’ or ‘Fred’s folly’) in which the preferred to call them) was initiated by the Italian crystallographers
model was optically superimposed onto the electron density by in 1976 at Erice, a medieval hilltop town in Sicily. These meetings
reflection of the model in a half-silvered mirror (Richards, 1968). have since been repeated every six years. The local organizer was
This allowed for convenient fitting of model parts and accurate Lodovico Riva di Sanseverino, whose vivacious sensitivity instilled
placement of atoms within the electron density. The Richards box a feeling of international fellowship into the rapidly growing
was the forerunner of today’s computer graphics, originally referred number of structural biologists. The first meeting lasted two whole
to as an ‘electronic Richards box’. The development of computer weeks, a length of time that would no longer be acceptable in
graphics for model building was initially met with reservation, but today’s hectic, competitive atmosphere.
fortunately those involved in these developments persevered. It took time for the staid organizers of the IUCr triennial congress
Various programs became available for model building in a to recognize the significance of macromolecular structure. Thus, for
computer, but the undoubted champion of this technology was many years, the IUCr meetings were poorly attended by structural
FRODO, written by Alwyn Jones (Jones, 1978). biologists. However, recent meetings have changed, with biological
topics representing about half of all activities. Nevertheless, the size
of these meetings and their lack of focus have led to numerous large
1.2.7. Meetings and small specialized meetings, from small Gordon Conferences
and East and West Coast crystallography meetings in the USA to
The birth of protein crystallography in the 1950s coincided with the
huge international congresses in virology, biochemistry and other
start of the jet age, making attendance at international meetings far
sciences.
easier. Indeed, the number and variety of meetings have proliferated
The publication of this volume by the International Union of
as much as the number and variety of structures determined. A
Crystallography, the first volume of International Tables devoted to
critical first for protein crystallography was a meeting held at the
macromolecular crystallography, strongly attests to the increasing
Hirschegg ski resort in Austria in 1966. This was organized by Max
importance of this vital area of science.
Perutz (Cambridge) and Walter Hoppe (München). About 40
scientists from around the world attended, as well as a strong
representation of students (including Robert Huber) from the
Acknowledgements
Münich laboratory. The first Hirschegg meeting occurred just
after the structure determination of lysozyme, which helped lift the I am gratefully indebted to Sharon Wilder, who has done much of
cloud of pessimism after the long wait for a new structure since the the background checking that was required to write this chapter.
structures of the globins had been solved in the 1950s. The meeting Furthermore, she has been my permanent and faithful helper during
was very much a family affair where most attendees stayed an extra a time that is, in part, covered in the review. I am additionally
few days for additional skiing. The times were more relaxed in indebted to Max Perutz and David Davies, both of whom read the
comparison with those of today’s jet-setting scientists flying manuscript very carefully, making it possible to add a few personal
directly from synchrotron to international meeting, making ever accounts. I also thank the National Institutes of Health and the
more numerous important discoveries. A second Hirschegg meeting National Science Foundation for generous financial support.
references
1.3. Macromolecular crystallography and medicine

BY W. G. J. HOL AND C. L. M. J. VERLINDE
1.3.1. Introduction (g) Chemistry, in particular combinatorial chemistry: discover-

ing by more and more sophisticated procedures high affinity
In the last hundred years, crystallography has contributed
inhibitors or binders to drug target proteins which are of great
immensely to the expansion of our understanding of the atomic
interest by themselves, while in addition such compounds tend to
structure of matter as it extends into the three spatial dimensions in
improve co-crystallization results quite significantly.
which we describe the world around us. At the beginning of this
(h) Crystallography itself: constantly developing new tools
century, the first atomic arrangements in salts, minerals and low-
including direct methods, multi-wavelength anomalous-dispersion
molecular-weight organic and metallo-organic compounds were
phasing techniques, maximum-likelihood procedures in phase
unravelled. Then, initially one by one, but presently as an
calculation and coordinate refinement, interactive graphics and
avalanche, the molecules of life were revealed in full glory at the
automatic model-building programs, density-modification methods,
atomic level with often astonishing accuracy, beginning in the
and the extremely important cryo-cooling techniques for protein
1950s when fibre diffraction first helped to resolve the structure of
and nucleic acid crystals, to mention only some of the major
DNA, later the structures of polysaccharides, fibrous proteins,
achievements in the last decade.
muscle and filamentous viruses. Subsequently, single-crystal
Numerous aspects of these developments are treated in great
methods became predominant and structures solved in the 1960s
detail in this volume of International Tables.
included myoglobin, haemoglobin and lysozyme, all of which were
heroic achievements by teams of scientists, often building their own
X-ray instruments, pioneering computational methods, and improv-
ing protein purification and crystallization procedures. Quite soon 1.3.2. Crystallography and medicine
thereafter, in 1978, the three-dimensional structures of the first Knowledge of accurate atomic structures of small molecules, such
viruses were determined at atomic resolution. Less than ten years as vitamin B12 , steroids, folates and many others, has assisted
later, the mechanisms and structures of membrane proteins started medicinal chemists in their endeavours to modify many of these
to be unravelled. Presently, somewhere between five and ten molecules for the combat of disease. The early protein crystal-
structures of proteins are solved each day, about 85% by lographers were well aware of the potential medical implication of
crystallographic procedures and about 15% by NMR methods. It the proteins they studied. Examples are the studies of the oxygen-
is quite possible that within a decade the Protein Data Bank (PDB; carrying haemoglobin, the messenger insulin, the defending
Bernstein et al., 1977) will receive a new coordinate set for a antibodies and the bacterial-cell-wall-lysing lysozyme. Yet, even
protein, RNA or DNA crystal structure every half hour. The by the mid-1980s, there were very few crystallographic projects
resolution of protein crystal structures is improving dramatically which had the explicit goal of arriving at pharmaceutically active
and the size of the structures tackled is sometimes enormous: a virus compounds (Hol, 1986). Since then, however, we have witnessed an
with over a thousand subunits has been solved at atomic resolution incredible increase in the number of projects in this area with
(Grimes et al., 1995) and the structure of the ribosome is on its way essentially every major pharmaceutical company having a protein
(Ban et al., 1999; Cate et al., 1999; Clemons et al., 1999). crystallography unit, while in academia and research institutions the
Macromolecular crystallography, discussed here in terms of its potential usefulness of a protein structure is often combined with
impact on medicine, is clearly making immense strides owing to a the novelty of the system under investigation. In one case, the HIV
synergism of progress in many scientific disciplines including: protease, it might well be that, worldwide, the structure has been
(a) Computer hardware and software: providing unprecedented solved over one thousand times – in complex with hundreds of
computer power as well as instant access to information anywhere different inhibitory compounds (Vondrasek et al., 1997).
on the planet via the internet. Impressive as these achievements are, this seems to be only the
(b) Physics: making synchrotron radiation available with a wide beginning of medicinal macromolecular crystallography. The
range of wavelengths, very narrow bandwidths and very high completion of the human genome project will provide an irresistible
intensities. impetus for ‘human structural genomics’: the determination, as
(c) Materials science and instrumentation: revolutionizing X-ray rapidly and systematically as possible, of as many human protein
intensity measurements, with currently available charge-coupled- structures as possible. The genome sequences of most major
device detectors allowing protein-data collection at synchrotrons in infectious agents will be completed five years hence, if not sooner.
tens of minutes, and with pixel array detectors on the horizon which This is likely to be followed up by ‘selected pathogen structural
are expected to collect a complete data set from a typical protein genomics’, which will provide a wealth of pathogen protein
within a few seconds. structures for the design of new pharmaceuticals and probably
(d ) Molecular biology: allowing the cloning, overexpression and also for vaccines.
modification of genes, with almost miraculous ease in many cases, This overview, written in late 1999, aims to convey some feel of
resulting in a wide variety of protein variants, thereby enabling the current explosion of ‘crystallography in medicine’. Ten, perhaps
crystallization of ‘impossible’ proteins. even five, years ago it might have been feasible to make an almost
(e) Genome sequencing: determining complete bacterial gen- comprehensive list of all protein structures of potentially direct
omes in a matter of months. With several eukaryote genomes and medical relevance. Today, this is virtually impossible. Here we
the first animal genome already completed, and with the human mention only selected examples in the text with apologies to the
genome expected to be completed to a considerable degree by 2000, crystallographers whose projects should also have been mentioned,
protein crystallographers suddenly have an unprecedented choice of and to the NMR spectroscopists and electron microscopists whose
proteins to study, giving rise to the new field of structural genomics. work falls outside the scope of this review. Tables 1.3.3.1 and
( f ) Biochemistry and biophysics: providing a range of tools for 1.3.4.1 to 1.3.4.5 provide more information, yet do not claim to
rapid protein and nucleic acid purification by size, charge and cover comprehensively this exploding field. Also, not all of the
affinity, and for characterization of samples by microsequencing, structures listed were determined with medical applications in
fluorescence, mass spectrometry, circular dichroism and dynamic mind, though they might be exploited for drug design one day.
light scattering procedures. These tables show at the same time tremendous achievements as
10

1.3. MACROMOLECULAR CRYSTALLOGRAPHY AND MEDICINE
well as great gaps in our structural knowledge of proteins from Hopkins University (Baltimore, MD) and the National Center for
humans and human pathogens. Biotechnology Information, National Library of Medicine
(Bethesda, MD), 1999. URL: http://www.ncbi.nlm.nih.gov/omim/],
with many more discoveries to occur in the next decades.
Biomolecular crystallography has been very successful in explain-
1.3.3. Crystallography and genetic diseases
ing the cause of numerous genetic diseases at the atomic level. The
Presently, an immense number of genetic diseases have been stories of sickle cell anaemia, thalassemias and other deficiencies of
characterized at the genetic level and archived in OMIM [On-line haemoglobin set the stage (Dickerson & Geis, 1983), followed by
Mendelian Inheritance in Man. Center for Medical Genetics, Johns numerous other examples (Table 1.3.3.1). Given the frequent
Table 1.3.3.1. Crystal structures and genetic diseases
Crystal structure Disease Reference
Acidic fibroblast growth factor receptor Familial Pfeiffer syndrome [1]

Alpha-1-antitrypsin Alpha-1-antitrypsin deficiency [2]
Antithrombin III Hereditary thrombophilia [3], [4]
Arylsulfatase A Leukodystrophy [5]
Aspartylglucosaminidase Aspartylglusominuria [6]
Beta-glucuronidase Sly syndrome [7]
Branched-chain alpha-keto acid dehydrogenase Maple syrup urine syndrome, type Ia [39]
Carbonic anhydrase II Guibaud–Vainsel syndrome, Marble brain disease [8]
p53 Cancer [9], [10]
Ceruloplasmin Hypoceruloplasminemia [11]
Complement C3 C3 complement component 3 deficiency [12]
Cystatin B Progressive myoclonus epilepsy [13]
Factor VII Factor VII deficiency [14]
Factor VIII Factor VIII deficiency [40]
Factor X Factor X deficiency (Stuart–Prower factor deficiency) [15]
Factor XIII Factor XIII deficiency [16]
Fructose-1,6-bisphosphate aldolase Fructose intolerance (fructosemia) [41]
Gelsolin Amyloidosis V [17]
Growth hormone Growth hormone deficiency [18]
Haemochromatosis protein HFE Hereditary haemochromatosis [19]
Haemoglobin Beta-thalassemia, sickle-cell anaemia [20]
Tyrosine hydroxylase Hereditary Parkinsonism [21]
Hypoxanthine–guanine phosphoribosyltransferase Lesch–Nyhan syndrome [22]
Insulin Hyperproinsulinemia, diabetes [42]
Isovaleryl–coenzyme A dehydrogenase Isovaleric acid CoA dehydrogenase deficiency [23]
Lysosomal protective protein Galactosialidosis [24]
Ornithine aminotransferase Ornithine aminotransferase deficiency [25]
Ornithine transcarbamoylase Ornithine transcarbamoylase deficiency [43]
p16INK4a tumour suppressor Cancer [26]
Phenylalanine hydroxylase Phenylketonuria [27]
Plasminogen Plasminogen deficiency [28], [29], [30]
Protein C Protein C deficiency [31]
Purine nucleotide phosphorylase Purine nucleotide phosphorylase deficiency [32]
Serum albumin Dysalbuminemic hyperthyroxinemia [33]
Superoxide dismutase (Cu, Zn-dependent) Familial amyotrophical lateral sclerosis [34]
Thrombin Hypoprothrombinemia, dysprothrombinemia [35]
Transthyretrin Amyloidosis I [36]
Triosephosphate isomerase Triosephosphate isomerase deficiency [37]
Trypsinogen Hereditary pancreatitis [38]
References: [1] Blaber et al. (1996); [2] Loebermann et al. (1984); [3] Carrell et al. (1994); [4] Schreuder et al. (1994); [5] Lukatela et al. (1998); [6] Oinonen et al.
(1995); [7] Jain et al. (1996); [8] Liljas et al. (1972); [9] Cho et al. (1994); [10] Gorina & Pavletich (1996); [11] Zaitseva et al. (1996); [12] Nagar et al. (1998); [13]
Stubbs et al. (1990); [14] Banner et al. (1996); [15] Padmanabhan et al. (1993); [16] Yee et al. (1994); [17] McLaughlin et al. (1993); [18] DeVos et al. (1992);
[19] Lebron et al. (1998); [20] Harrington et al. (1997); [21] Goodwill et al. (1997); [22] Eads et al. (1994); [23] Tiffany et al. (1997); [24] Rudenko et al. (1995);
[25] Shah et al. (1997); [26] Russo et al. (1998); [27] Erlandsen et al. (1997); [28] Mulichak et al. (1991); [29] Mathews et al. (1996); [30] Chang, Mochalkin et al.
(1998); [31] Mather et al. (1996); [32] Ealick et al. (1990); [33] He & Carter (1992); [34] Parge et al. (1992); [35] Bode et al. (1989); [36] Blake et al. (1978); [37]
Mande et al. (1994); [38] Gaboriaud et al. (1996); [39] Ævarsson et al. (2000); [40] Pratt et al. (1999); [41] Gamblin et al. (1990); [42] Bentley et al. (1976); [43]
Shi et al. (1998).
11
1. INTRODUCTION
occurrence of mutations in humans, it is likely that for virtually al., 1986), an enzyme responsible for much of the cellular damage
every structure of a human protein, a number of genetic diseases associated with cystic fibrosis (Birrer, 1995). On the basis of the
can be rationalized at the atomic level. Two investigations from the elastase structure, inhibitors were developed to combat the effects
authors’ laboratory may serve as examples: of the impaired ion channel (Warner et al., 1994). Also, structures
(i) The severity of various cases of galactosialidosis – a of key enzymes of Pseudomonas aeruginosa, a bacterium affecting
lysosomal storage disease – could be related to the predicted effects many cystic fibrosis patients, form a basis for the design of
of the amino-acid substitutions on the stability of human protective therapeutics to treat infections by this pathogen. Yet, to the best of
protein cathepsin A (Rudenko et al., 1998). our knowledge, no compound has been developed so far that repairs
(ii) The modification of Tyr393 to Asn in the branched-chain the malfunctioning ion channel.
2-oxo acid dehydrogenase occurs at the interface of the and However, in some cases there might be more opportunities than
subunits in this 2 2 heterotetramer, providing a nice explanation of assumed so far. Several mutations leading to genetic diseases result
the ‘mennonite’ variants of maple syrup urine disease (MSUD) in a lack of stability of the affected protein. In instances when the
(Ævarsson et al., 2000). mutant protein is still stable enough to fold, small molecules could
Impressive as the insights obtained into the causes of diseases conceivably be discovered that bind ‘anywhere’ to a pocket of these
like these might be, there is almost a sense of tragedy associated proteins, thereby stabilizing the protein. The same small molecule
with this detailed understanding of a serious, sometimes fatal, could even be able to increase the stability of proteins with different
afflictions at the atomic and three-dimensional level: there is often mildly destabilizing mutations. Such an approach, though not trivial
so little one can do with this knowledge. There are at least two, very by any means, might be worth pursuing. Proof of principle of this
different, reasons for this. The first reason is that turning a concept has recently been provided for several unstable p53
malfunctioning protein or nucleic acid into one that functions mutants, where the same small molecule enhanced the stability of
properly is notoriously difficult. Treatment would generally require different mutants (Foster et al., 1999)
the oral use of small molecules that somehow counteract the effect Of course, mutations that destroy cofactor binding or active sites,
of the mutation, i.e. the administration of the small molecule has to or destroy proper recognition of partner proteins, will be extremely
result in a functional complex of the drug with the mutant protein. difficult to correct by small molecules targeting the affected protein.
This is in almost all cases far more difficult than finding compounds In such instances, gene therapy is likely to be the way by which our
that block the activity of a protein or nucleic acid – which is the way and the next generation may be able to improve the lives of future
in which most current drugs function. The second reason for the generations.
paucity of drugs for treating genetic diseases is very different in
nature: the number of patients suffering from a particular mutation
responsible for a genetic disease is very small in most cases. This
1.3.4. Crystallography and development of novel
means that market forces do not encourage funding the expensive
pharmaceuticals
steps of testing the toxicity and efficacy of potentially pharmaceu-
tically active compounds. One of several exceptions is sickle cell The impact of detailed knowledge of protein and nucleic acid
anaemia, where significant efforts have been made to arrive at structures on the design of new drugs has already been significant,
pharmaceutically active agents (Rolan et al., 1993). In this case the and promises to be of tremendous importance in the next decades.
mutation Glu6 Val leads to deoxyhaemoglobin polymerization via The first structure of a known major drug bound to a target protein
the hydrophobic valine. In spite of several ingenious approaches was probably that of methotrexate bound to dihydrofolate reductase
based on the allosteric properties of haemoglobin (Wireko & (DHFR) (Matthews et al., 1977). Even though the source of the
Abraham, 1991), no successful compound seems to be on the enzyme was bacterial while methotrexate is used as a human
horizon yet for the treatment of sickle cell anaemia. anticancer agent, this protein–drug complex structure was never-
More recently, the spectacular molecular mechanisms underlying theless a hallmark achievement. It is generally accepted that the first
genetic serpin deficiency diseases have been elucidated. A typical protein-structure-inspired drug actually reaching the market was
example is 1-antitrypsin deficiency, which leads to cirrhosis and captopril, which is an antihypertensive compound blocking the
emphysema. Normal 1-antitrypsin, a serine protease inhibitor, action of angiotensin-converting enzyme, a metalloprotease. In this
exposes a peptide loop as a substrate for the cognate proteinase in its case, the structure of zinc-containing carboxypeptidase A was a
active but metastable conformation. After cleavage of the loop, the guide to certain aspects of the chemical modification of lead
protease becomes trapped as an acyl-enzyme with the serpin, and compounds (Cushman & Ondetti, 1991). This success has been
the cleaved serpin loop inserts itself as the central strand of one of followed up by numerous projects specifically aimed at the design
the serpin -sheets, accompanied by a dramatic change in protein of new inhibitors, or activators, of carefully selected drug targets.
stability. In certain mutant serpins, however, the exposed loop is Structure-based drug design (SBDD) (Fig. 1.3.4.1) is the subject
conformationally more metastable and occasionally inserts itself of several books and reviews that summarize projects and several
into the -sheet of a neighbouring serpin molecule, thereby forming success stories up until the mid-1990s (Kuntz, 1992; Perutz, 1992;
serpin polymers with disastrous consequences for the patient Verlinde & Hol, 1994; Whittle & Blundell, 1994; Charifson, 1997;
(Carrell & Gooptu, 1998). In vitro, the polymerization of 1- Veerapandian, 1997). Possibly the most dramatic impact made by
antitrypsin can be reversed with synthetic homologues of the SBDD has been on the treatment of AIDS, where the development
exposed peptide loop (Skinner et al., 1998). This approach might be of essentially all of the protease inhibitors on the market in 1999 has
useful for other ‘conformational diseases’, which include Alzhei- been guided by, or at least assisted by, the availability of numerous
mer’s and other neurodegenerative disorders. crystal structures of protease–inhibitor complexes.
Another frequently occurring genetic disease is cystic fibrosis. The need for a large number of structures is common in all drug
Here we face a more complex situation than that in the case of sickle design projects and is due to several factors. One is the tremendous
cell anaemia: a range of different mutations causes a malfunctioning challenge for theoretical predictions of the correct binding mode
of the same ion channel, which, consequently, leads to a range of and affinity of inhibitors to proteins. The current force fields are
severity of the disease (Collins, 1992). Protein crystallography is approximate, the properties of water are treacherous, the flexibility
currently helpful in an indirect way in alleviating the problems of of protein and ligands lead quickly to a combinatorial explosion,
cystic fibrosis patients, not by studying the affected ion channel and the free-energy differences between various binding modes are
itself, but by revealing the structure of leukocyte elastase (Bode et small. All this leads to the need for several experimental structures
12
in a structure-based drug design cycle (Fig. 1.3.4.1). In this cycle, providing platforms on the basis of which the design of novel drugs
numerous disciplines are interacting in multiple ways. Many is actively pursued (Le et al., 1998).
institutions, small and large, are following in one way or another A quite spectacular example of how structural knowledge can
this paradigm to speed up the lead discovery, lead optimization and lead to the synthesis of powerful inhibitors is provided by influenza
even the bioavailability improvement steps in the drug development virus neuraminidase. The structure of a neuraminidase–transition-
process. Moreover, a very powerful synergism exists between state analogue complex suggested the addition of positively charged
combinatorial chemistry and structure-based drug design. Struc- amino and guanidinium groups to the C4 position of the analogue,
ture-guided combinatorial libraries can utilize knowledge of ligand which resulted, in one step, in a gain of four orders of magnitude in
target sites in the design of the library [see e.g. Ferrer et al. (1999), binding affinity for the target enzyme (von Itzstein et al., 1993).
Eckert et al. (1999) and Minke et al. (1999)]. Once tight-binding
ligands are found by combinatorial methods, crystal structures of 1.3.4.1.2. Bacterial diseases
library compound–target complexes provide detailed information
A very large number of structures of important drug target
for new highly specific libraries.
proteins of bacterial origin have been solved crystallographically
The fate of a drug candidate during clinical tests can hinge on a
(Table 1.3.4.2). Currently, the most important single infectious
single methyl group – just as a point mutation can alter the benefit of
bacterial agent is Mycobacterium tuberculosis, with three million
a wild-type protein molecule into the nightmare of a life-long
deaths and eight million new cases annually (Murray & Salomon,
genetic disease. Hence, many promising inhibitors eventually fail to
1998). The crystal structures of several M. tuberculosis potential
be of benefit to patients. Nevertheless, knowledge of a series of
and proven drug target proteins have been elucidated (Table
protein structures in complex with inhibitors is of immense value in
1.3.4.2). The complete M. tuberculosis genome has been sequenced
the design and development of future pharmaceuticals. In the
recently (Cole et al., 1998), and this will undoubtedly have a
following sections some examples will be looked at.
tremendous impact on future drug development.
The crystal structures of many bacterial dihydrofolate reductases,
the target of several antimicrobials including trimethoprim, have
1.3.4.1. Infectious diseases also been reported. Recently, the atomic structure of dihydroptero-
ate synthase (DHPS), the target of sulfa drugs, has been elucidated,
1.3.4.1.1. Viral diseases almost 60 years after the first sulfa drugs were used to treat patients
Some icosahedral pathogenic viruses have all their capsid (Achari et al., 1997; Hampele et al., 1997).
proteins elucidated, while for the more complex viruses like A very special set of bacterial proteins are the toxins. Some of
influenza virus, hepatitis C virus (HCV) and HIV, numerous these have dramatic effects, with even a single molecule able to kill
individual protein structures have been solved (Table 1.3.4.1). a host cell. These toxins outsmart and (mis)use many of the defence
However, not all 14 native proteins of the HIV genome have yet systems of the host, and their structures are often most unusual and
surrendered to the crystallographic community, nor to the NMR fascinating, as recently reviewed by Lacy & Stevens (1998). The
spectroscopists or the high-resolution electron microscopists, our structures of the toxins are actively used for the design of
partners in experimental structural biology (Turner & Summers, prophylactics and therapeutic agents to treat bacterial diseases
1999). Nevertheless, the structures of HIV protease, reverse [see e.g. Merritt et al. (1997), Kitov et al. (2000) and Fan et al.
transcriptase and fragments of HIV integrase and of HIV viral (2000)]. It is remarkable that the properties of these devastating
core and surface proteins are of tremendous value for developing toxins are also used, or at least considered, for the treatment of
novel anti-AIDS therapeutics [Arnold et al., 1996; Lin et al., 1998; disease, such as in the engineering of diphtheria toxin to create
Wlodawer & Vondrasek, 1998; see also references in Table immunotoxins for the treatment of cancer and the deployment of
1.3.4.1(a)]. A similar situation occurs for hepatitis C virus. The cholera toxin mutants as adjuvants in mucosal vaccines. Knowledge
protease structure of this virus has been solved recently of the three-dimensional structures of these toxins assists in the
(simultaneously by four groups!), as well as its helicase structure, design of new therapeutically useful proteins.
1.3.4.1.3. Protozoan infections

A major cause of death and worldwide
suffering is due to infections by several
protozoa, including:
(a) Plasmodium falciparum and related
species, causing various forms of malaria;
(b) Trypanosoma cruzi, the causative
agent of Chagas’ disease in Latin America;
(c) Trypanosoma brucei, causing sleep-
ing sickness in Africa;
(d) Some eleven different Leishmania
species, responsible for several of the most
horribly disfiguring diseases known to
mankind.
Drug resistance, combined with other
factors, has been the cause of a major
disappointment for the early hopes of a
‘malaria eradication campaign’. Fortu-
nately, new initiatives have been launched
recently under the umbrella of the ‘Malaria
roll back’ program and the ‘Multilateral
Fig. 1.3.4.1. The structure-based drug design cycle. Initiative for Malaria’ (MIM). We are
13
1. INTRODUCTION
Table 1.3.4.1. Important human pathogenic viruses and their proteins
(a) RNA viruses
(i) Single-stranded
Family Example Protein structures solved Reference
Arenaviridae Lassa fever virus None

Bunyaviridae Hantavirus None
Caliciviridae Hepatitis E virus, Norwalk virus None
Coronaviridae Corona virus None
Deltaviridae Hepatitis D virus Oligomerization domain of antigen [1]
Filoviridae Ebola virus GP2 of membrane fusion glycoprotein [2]
Flaviviridae Dengue NS3 protease [3]
Hepatitis C NS3 protease [4], [5]
RNA helicase [6]
Yellow fever None
Tick-borne encephalitis virus Envelope glycoprotein [7]
Orthomyxoviridae Influenza virus Neuraminidase [8]
Haemagglutinin [9]
Matrix protein M1 [10]
Paramyxoviridae Measles, mumps, parainfluenza, respiratory None
syncytial virus
Picornaviridae Hepatitis A virus 3C protease [11]
Poliovirus Capsid [12]
RNA-dependent polymerase [13]
Rhinovirus Capsid [14]
3C protease [15]
Echovirus Capsid [16]
Retroviridae HIV Capsid protein [17]
Matrix protein [18]
Protease [19], [20], [21]
Reverse transcriptase [22], [23], [47], [48], [49]
Integrase [24]
gp120 [25]
NEF [26]
gp41 [27]
Rhabdovirus Rabies virus None
Togaviridae Rubella None
(ii) Double-stranded

Reoviridae Rotavirus None
(b) DNA viruses

(i) Single-stranded

Parvoviridae B 19 virus None
(ii) Double-stranded

Adenoviridae Adenovirus Protease [28]
Capsid [29]
Knob domain of fibre protein [30]
Hepadnaviridae Hepatitis B Capsid [31]
Herpesviridae Cytomegalovirus Protease [32], [33], [34]
Epstein–Barr virus Domains of nuclear antigen 1 [35]
BCRF1 [36]
14
Table 1.3.4.1. Important human pathogenic viruses and their proteins (cont.)

Herpes simplex Protease [37]
Thymidine kinase [38]
Uracyl-DNA glycosylase [39]
Core of VP16 [40]
Varicella zoster Protease [42]
Papovaviridae Papillomavirus DNA-binding domain of E2 [43]
Activation domain of E2 [44]
Poxviridae Smallpox virus None
Vaccinia virus (related to smallpox but non- Methyltransferase VP39 [45]
pathogenic) Domain of topoisomerase [46]
References: [1] Zuccola et al. (1998); [2] Weissenhorn et al. (1998); [3] Murthy et al. (1999); [4] Love et al. (1996); [5] Yan et al. (1998); [6] Yao et al. (1997); [7]
Rey et al. (1995); [8] Varghese et al. (1983); [9] Wilson et al. (1981); [10] Sha & Luo (1997); [11] Allaire et al. (1994); [12] Hogle et al. (1985); [13] Hansen et al.
(1997); [14] Rossmann et al. (1985); [15] Matthews et al. (1994); [16] Filman et al. (1998); [17] Worthylake et al. (1999); [18] Hill et al. (1996); [19] Navia,
Fitzgerald et al. (1989); [20] Wlodawer et al. (1989); [21] Erickson et al. (1990); [22] Rodgers et al. (1995); [23] Ding et al. (1995); [24] Dyda et al. (1994); [25]
Kwong et al. (1998); [26] Lee et al. (1996); [27] Chan et al. (1997); [28] Ding et al. (1996); [29] Roberts et al. (1986); [30] Xia et al. (1994); [31] Wynne et al.
(1999); [32] Tong et al. (1996); [33] Qiu et al. (1996); [34] Shieh et al. (1996); [35] Bochkarev et al. (1995); [36] Zdanov et al. (1997); [37] Hoog et al. (1997);
[38] Wild et al. (1995); [39] Savva et al. (1995); [40] Liu et al. (1999); [42] Qiu et al. (1997); [43] Hegde & Androphy (1998); [44] Harris & Botchan (1999); [45]
Hodel et al. (1996); [46] Sharma et al. (1994); [47] Kohlstaedt et al. (1992); [48] Jacobo-Molina et al. (1993); [49] Ren et al. (1995).
facing a formidable challenge, however, since the parasite is very disease, even though it does not kill the adult worms. Therefore, a
clever at evading the immune response of the human host. Drugs are biological clock is ticking, waiting until resistance occurs against
the mainstay of current treatments and may well be so for a long this single compound available for treatment. Schistosoma species
time to come. Protein crystallographic studies of Plasmodium are responsible for a wide variety of liver diseases and are spreading
proteins are hampered by the unusual codon usage of the continuously since irrigation schemes provide a perfect environ-
Plasmodium species, coupled with a tendency to contain insertions ment for the intermediate snail vector. Other medically important
of numerous hydrophilic residues in the polypeptide chain (Gardner helminths are Wuchereria bancrofti and Brugia malayi. Only a few
et al., 1998) which provide sometimes serious obstacles to protein structures from these very important disease-causing
obtaining large amounts of Plasmodium proteins for structural organisms have been unravelled so far (Table 1.3.4.3).
investigations.
However, the structures of an increasing number of potential
1.3.4.2. Resistance
drug targets from these protozoan parasites are being unravelled.
These include the variable surface glycoproteins (VSGs) and Resistance to drugs in infectious organisms, as well as in cancers,
glycolytic enzymes of Trypanosoma brucei, crucial malaria is a fascinating subject, since it demonstrates the action and reaction
proteases, and the remarkable trypanothione reductase (Table of biological systems in response to environmental challenges. Life,
1.3.4.3). The structures of nucleotide phosphoribosyl transferases of course, has been evolving to do just that – and the arrival of new
of a variety of protozoan parasites have also been elucidated. chemicals, termed ‘drugs’, on the scene is nothing new to organisms
Moreover, the structure of DHFR from Pneumocystis carinii, the that are the result of evolutionary processes involving billions of
major opportunistic pathogen in AIDS patients in the United States, years of chemical warfare. Populations of organisms span a wide
has been determined. Several of these structures are serving as range of variation at the genetic and protein levels, and the chance
starting points for the development of new drugs. that one of the variants is able to cope with drug pressure is nonzero.
The variety of mechanisms observed to be responsible for drug
1.3.4.1.4. Fungi resistance is impressive (Table 1.3.4.4).
Crystallography has made major contributions to the detailed
In general, the human immune system is able to keep the growth
molecular understanding of resistance in the case of detoxification,
of fungi under control, but in immuno-compromised patients (e.g.
mutation and enzyme replacement mechanisms. Splendid examples
as a result of cancer chemotherapy, HIV infection, transplantation
are:
patients receiving immunosuppressive drugs, genetic disorders)
(a) The beta-lactamases: These beta-lactam degrading enzymes,
such organisms are given opportunities they usually do not have.
of which there are four classes, are produced by many bacteria to
Candida albicans is an opportunistic fungal organism which causes
counteract penicillins and cephalosporins, the most widely used
serious complications in immuno-compromised patients. Several of
antibiotics on the planet. No less than 53 structures of these
its proteins have been structurally characterized (Table 1.3.4.3) and
enzymes reside in the PDB.
provide opportunities for the development of selectively active
(b) HIV protease mutations: Tens of mutations have been
inhibitors.
characterized structurally. Many alter the active site at the site of
mutation, thereby preventing drug binding. Other mutations
1.3.4.1.5. Helminths
rearrange the protein backbone, reshaping entire pockets in the
Worms affect the lives of billions of human beings, causing binding site (Erickson & Burt, 1996).
serious morbidity in many ways. Onchocerca volvolus is the cause (c) HIV reverse transcriptase mutations: Via structural studies, at
of river blindness, which resulted in the virtual disappearance of least three mechanisms of drug resistance have been elucidated:
entire villages in West Africa, until ivermectin appeared. This direct alteration of the binding sites for the nucleoside analogue or
remarkable compound dramatically reduced the incidence of the non-nucleoside inhibitors, mutations that change the position of the
15
1. INTRODUCTION
Table 1.3.4.2. Protein structures of important human pathogenic bacteria
Organism Disease(s) Protein structures solved Reference

Staphylococcus aureus Abscesses Alpha-haemolysin [1]
Endocarditis Aureolysin [2]
Gastroenteritis Beta-lactamase [3]
Toxic shock syndrome Collagen adhesin [4]
7,8-Dihydroneopterin aldolase [5]
Dihydropteroate synthetase [6]
Enterotoxin A [7]
Enterotoxin B [8]
Enterotoxin C2 [9]
Enterotoxin C3 [10]
Exfoliative toxin A [11]
Ile-tRNA-synthetase [12]
Kanamycin nucleotidyltransferase [13]
Leukocidin F [14]
Nuclease [15]
Staphopain [16]
Staphylokinase [17]
Toxic shock syndrome toxin-1 [18]
Staphylococcus epidermidis Implant infections None
Enterococcus faecalis Urinary tract and biliary tract infections NADH peroxidase [19]
(Streptococcus faecalis) Histidine-containing phosphocarrier protein [20]
Streptococcus mutans Endocarditis Glyceraldehyde-3-phosphate dehydrogenase [21]
Streptococcus pneumoniae Pneumonia Penicillin-binding protein PBP2x [22]
Meningitis, upper respiratory tract infections Dpnm DNA adenine methyltransferase [23]
Streptococcus pyogenes Pharyngitis Inosine monophosphate dehydrogenase [24]
Scarlet fever, toxic shock syndrome, immunologic Pyrogenic exotoxin C [25]
disorders (acute glomerulonephritis and
rheumatic fever)
Bacillus anthracis Anthrax Anthrax protective antigen [26]
Bacillus cereus Food poisoning Beta-amylase [27]
Beta-lactamase II [28]
Neutral protease [29]
Oligo-1,6-glucosidase [30]
Phospholipase C [31]
Clostridium botulinum Botulism Neurotoxin type A [32]
Clostridium difficile Pseudomembranous colitis None
Clostridium perfringens Gas gangrene Alpha toxin [33]
Food poisoning Perfringolysin O [34]
Clostridium tetani Tetanus Toxin C fragment [35]
Corynebacterium Diphtheria Toxin [36]
diphtheriae Toxin repressor [37]
Listeria monocytogenes Meningitis, sepsis Phosphatidylinositol-specific phospholipase C [38]

Actinomyces israelii Actinomycosis None
Nocardia asteroides Nocardiosis None
Neisseria gonorrhoeae Gonorrhea Type 4 pilin [39]
Carbonic anhydrase [40]
Neisseria meningitidis Meningitis Dihydrolipoamide dehydrogenase [41]
Bordetella pertussis Whooping cough Toxin [42]
Virulence factor P.69 [43]
Brucella sp. Brucellosis None
Campylobacter jejuni Enterocolitis None
Enterobacter cloacae Urinary tract infection, pneumonia Beta-lactamase: class C [44]
UDP-N-acetylglucosamine enolpyruvyltransferase [45]
Escherichia coli
ETEC (enterotoxigenic) Traveller’s diarrhoea Heat-labile enterotoxin [46]
Heat-stable enterotoxin (is a peptide) [47]
16
Table 1.3.4.2. Protein structures of important human pathogenic bacteria (cont.)

EHEC HUS Verotoxin-1 [48]
(enterohaemorrhagic)
EPEC (enteropathogenic) Diarrhoea None
EAEC (enteroaggregative) Diarrhoea None
EIEC (enteroinvasive) Diarrhoea None
UPEC (uropathogenic) FimH adhesin [49]
FimC chaperone [49]
PapD [50]
NMEC (neonatal Meningitis None
meningitis)
Franciscella tularensis Tularemia None
Haemophilus influenzae Meningitis, otitis media, pneumonia Diaminopimelate epimerase [51]
6-Hydroxymethyl-7,8-dihydropterin [52]
pyrophosphokinase
Ferric iron binding protein Mirp [53]
Klebsiella pneumoniae Urinary tract infection, pneumonia, sepsis -Lactamase SHV-1 [54]
Legionella pneumophila Pneumonia None
Pasteurella multocida Wound infection None
Proteus mirabilis Pneumonia, urinary tract infection Catalase [55]
Glutathione S-transferase [56]
Proteus vulgaris Urinary tract infections Pvu II DNA-(cytosine N4) methyltransferase [57]
Pvu II endonuclease [58]
Tryptophanase [59]
Salmonella typhi Typhoid fever None, but many for S. typhimurium linked with
zoonotic disease
Salmonella enteridis Enterocolitis None
Serratia marcescens Pneumonia, urinary tract infection Serralysin [60]
Aminoglycoside 3-N-acetyltransferase [61]
Chitinase A [62]
Chitobiase [63]
Endonuclease [64]
Hasa (haemophore) [65]
Prolyl aminopeptidase [66]
Shigella sp. Dysentery Chloramphenicol acetyltransferase [67]
Shiga-like toxin I [68]
Vibrio cholerae Cholera Cholera toxin [69], [70]
DSBA oxidoreductase [71]
Neuraminidase [104]
Yersinia enterocolitica Enterocolitis Protein-Tyr phosphatase YOPH [72]
Yersinia pestis Plague None
Pseudomonas aeruginosa Wound infection, urinary tract infection, Alkaline metalloprotease [73]
pneumonia, sepsis Amidase operon [74]
Azurin [75]
Cytochrome 551 [76]
Cytochrome c peroxidase [77]
Exotoxin A [78]
p-Hydroxybenzoate hydroxylase [79]
Hexapeptide xenobiotic acetyltransferase [80]
Mandelate racemase [81]
Nitrite reductase [82]
Ornithine transcarbamoylase [83]
Porphobilinogen synthase [84]
Pseudolysin [85]
Burkholderia cepacia Wound infection, urinary tract infection, Biphenyl-cleaving extradiol dioxygenase [86]
pneumonia, sepsis cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase [87]
Dialkylglycine decarboxylase [88]
Lipase [89]
17
1. INTRODUCTION
Table 1.3.4.2. Protein structures of important human pathogenic bacteria (cont.)

Phthalate dioxygenase reductase [90]
Stenotrophomonas Sepsis None
maltophilia
(= Pseudomonas
maltophilia)
Bacteroides fragilis Intra-abdominal infections Beta-lactamase type 2 [91]
Mycobacterium leprae Leprosy Chaperonin-10 (GroES homologue) [92]
RUVA protein [93]
Mycobacterium tuberculosis Tuberculosis 3-Dehydroquinate dehydratase [94]
Dihydrofolate reductase [95]
Dihydropteroate synthase [96]
Enoyl acyl-carrier-protein reductase (InhA) [97]
Mechanosensitive ion channel [98]
Quinolinate phosphoribosyltransferase [99]
Superoxide dismutase (iron dependent) [100]
Iron-dependent repressor
Mycobacterium bovis Tuberculosis Tetrahydrodipicolinate-N-succinyltransferase [102]
Chlamydia psitacci Psittacosis None
Chlamydia pneumoniae Atypical pneumonia None
Chlamydia trahomatis Ocular, respiratory and genital infections None
Coxiella burnetii Q fever None
Rickettsia sp. Rocky Mountain spotted fever None
Borrelia burgdorferi Lyme disease Outer surface protein A [103]
Leptospira interrogans Leptospirosis None
Treponema pallidum Syphilis None
Mycoplasma pneumoniae Atypical pneumonia None
References: [1] Song et al. (1996); [2] Banbula et al. (1998); [3] Herzberg & Moult (1987); [4] Symersky et al. (1997); [5] Hennig et al. (1998); [6] Hampele et al.
(1997); [7] Sundstrom et al. (1996); [8] Papageorgiou et al. (1998); [9] Papageorgiou et al. (1995); [10] Fields et al. (1996); [11] Vath et al. (1997); [12] Silvian et
al. (1999); [13] Pedersen et al. (1995); [14] Pedelacq et al. (1999); [15] Loll & Lattman (1989); [16] Hofmann et al. (1993); [17] Rabijns et al. (1997); [18] Prasad
et al. (1993); [19] Yeh et al. (1996); [20] Jia et al. (1993); [21] Cobessi et al. (1999); [22] Pares et al. (1996); [23] Tran et al. (1998); [24] Zhang, Evans et al.
(1999); [25] Roussel et al. (1997); [26] Petosa et al. (1997); [27] Mikami et al. (1999); [28] Carfi et al. (1995); [29] Pauptit et al. (1988); [30] Watanabe et al.
(1997); [31] Hough et al. (1989); [32] Lacy et al. (1998); [33] Naylor et al. (1998); [34] Rossjohn, Feil, McKinstry et al. (1997); [35] Umland et al. (1997); [36]
Choe et al. (1992); [37] Qiu et al. (1995); [38] Moser et al. (1997); [39] Parge et al. (1995); [40] Huang, Xue et al. (1998); [41] Li de la Sierra et al. (1997); [42]
Stein et al. (1994); [43] Emsley et al. (1996); [44] Lobkovsky et al. (1993); [45] Schonbrunn et al. (1996); [46] Sixma et al. (1991); [47] Ozaki et al. (1991); [48]
Stein et al. (1992); [49] Choudhury et al. (1999); [50] Sauer et al. (1999); [51] Cirilli et al. (1993); [52] Hennig et al. (1999); [53] Bruns et al. (1997); [54] Kuzin et
al. (1999); [55] Gouet et al. (1995); [56] Rossjohn, Polekhina et al. (1998); [57] Gong et al. (1997); [58] Athanasiadis et al. (1994); [59] Isupov et al. (1998); [60]
Baumann (1994); [61] Wolf et al. (1998); [62] Perrakis et al. (1994); [63] Tews et al. (1996); [64] Miller et al. (1994); [65] Arnoux et al. (1999); [66] Yoshimoto et
al. (1999); [67] Murray et al. (1995); [68] Ling et al. (1998); [69] Merritt et al. (1994); [70] Zhang et al. (1995); [71] Hu et al. (1997); [72] Stuckey et al. (1994);
[73] Miyatake et al. (1995); [74] Pearl et al. (1994); [75] Adman et al. (1978); [76] Almassy & Dickerson (1978); [77] Fulop et al. (1995); [78] Allured et al.
(1986); [79] Gatti et al. (1994); [80] Beaman et al. (1998); [81] Kallarakal et al. (1995); [82] Nurizzo et al. (1997); [83] Villeret et al. (1995); [84] Frankenberg et
al. (1999); [85] Thayer et al. (1991); [86] Han et al. (1995); [87] Hulsmeyer et al. (1998); [88] Toney et al. (1993); [89] Kim et al. (1997); [90] Correll et al. (1992);
[91] Concha et al. (1996); [92] Mande et al. (1996); [93] Roe et al. (1998); [94] Gourley et al. (1999); [95] Li et al. (2000); [96] Baca et al. (2000); [97] Dessen et
al. (1995); [98] Chang, Spencer et al. (1998); [99] Sharma et al. (1998); [100] Cooper et al. (1995); [102] Beaman et al. (1997); [103] Li et al. (1997); [104]
Crennell et al. (1994).
DNA template, and mutations that induce conformational changes et al., 1996). Thus far, the structures of vanX (Bussiere et al., 1998)
that propagate into the active site (Das et al., 1996; Hsiou et al., and D-Ala-D-Ala ligase as a model for vanA (Fan et al., 1994) have
1998; Huang, Chopra et al., 1998; Ren et al., 1998; Sarafianos et al., been solved. They provide an exciting basis for arriving at new
1999). antibiotics against vancomycin-resistant bacteria.
(d) Resistance to vancomycin: In non-resistant bacteria, (e) DHFR: Some bacteria resort to the ‘ultimate mutation’ in
vancomycin stalls the cell-wall synthesis by binding to the D-Ala- order to escape the detrimental effects of antibiotics. They simply
D-Ala terminus of the lipid–PP-disaccharide–pentapeptide substrate replace the entire targeted enzyme by a functionally identical but
of the bacterial transglycosylase/transpeptidase, thereby sterically structurally different enzyme. A prime example is the presence of a
preventing the approach of the substrate. Resistant bacteria, dimeric plasmid-encoded DHFR in certain trimethoprim-resistant
however, have acquired a plasmid-borne transposon encoding for bacteria. The structure proved to be unrelated to that of the
five genes, vanS, vanR, vanH, vanA and vanX, that allows them to chromosomally encoded monomeric DHFR (Narayana et al., 1995).
synthesise a substrate ending in D-Ala-D-lactate. This minute Clearly, the structural insight gained from these studies provides
difference, an oxygen atom replacing an NH, leads to a 1000-fold us with avenues towards methods for coping with the rapid and
reduced affinity for vancomycin, explaining the resistance (Walsh alarming spread of resistance against available antibiotics that
18
Table 1.3.4.3. Protein structures of important human pathogenic protozoa, fungi and helminths
(a) Protozoa
Organism Disease Protein structures solved Reference
Acanthamoeba sp. Opportunistic meningoencephalitis, corneal Actophorin [1]

ulcers Profilin [2]
Cryptosporidium parvum Cryptosporidiosis None
Entamoeba histolytica Amoebic dysentery, liver abscesses None
Giardia lamblia Giardiasis None
Leishmania sp. Leishmaniasis Adenine phosphoribosyltransferase [3]
Dihydrofolate reductase-thymidylate [4]
synthase
Glyceraldehyde-3-phosphate dehydrogenase [5]
Leishmanolysin [6]
Nucleoside hydrolase [7]
Pyruvate kinase [8]
Triosephosphate isomerase [9]
Plasmodium sp. Malaria Fructose-1,6-bisphosphate aldolase [10]
Lactate dehydrogenase [11]
MSP1 [12]
Plasmepsin II [13]
Purine phosphoribosyltransferase [14]
Pneumocystis carinii Pneumonia Dihydrofolate reductase [16]
Toxoplasma gondii Toxoplasmosis HGXPRTase [17]
UPRTase [18]
Trichomonas vaginalis Trichomoniasis None
Trypanosoma brucei Sleeping sickness Fructose-1,6-bisphosphate aldolase [19]
6-Phosphogluconate dehydrogenase [21]
Phosphoglycerate kinase [22]
VSG [24]
Trypanosoma cruzi Chagas’ disease Cruzain (cruzipain) [25]
Hypoxanthine phosphoribosyltransferase [27]
Trypanothione reductase [29]
Tyrosine aminotransferase [30]
(b) Fungi

Aspergillus fumigatus Aspergillosis Restrictocin [31]
Blastomyces dermatidis Blastomycosis None
Candida albicans Candidiasis Dihydrofolate reductase [32]
N-Myristoyl transferase [33]
Phosphomannose isomerase [34]
Secreted Asp protease [35]
Coccidiodes immitis Coccidioidomycosis None
Cryptococcus neoformans Cryptococcosis None
Histoplasma capsulatum Histoplasmosis None
Mucor sp. Mucormycosis None
Paracoccidioides brasiliensis Paracoccidioidomycosis None
Rhizopus sp. Phycomycosis Lipase II [36]
Rhizopuspepsin [37]
RNase Rh [38]
19
1. INTRODUCTION
Table 1.3.4.3. Protein structures of important human pathogenic protozoa, fungi and helminths (cont.)
(c) Helminths

Clonorchis sinensis Clonorchiasis None
Fasciola hepatica Fasciolasis Glutathione S-transferase [39]
Fasciolopsis buski Fasciolopsiasis None
Paragominus westermani Paragonimiasis None
Schistosoma sp. Schistosomiasis Glutathione S-transferase [39], [40]
Hexokinase [41]
Diphyllobotrium latum Diphyllobothriasis None
Echinococcus granulosus Unilocular hydatid cyst disease None
Taenia saginata Taeniasis None
Taenia solium Taeniasis None
Ancylostoma duodenale Old World hookworm disease None
Anisakis Anisakiasis None
Ascaris lumbricoides Ascariasis Haemoglobin [42]
Major sperm protein [43]
Trypsin inhibitor [44]
Enterobius vermicularis Pinworm infection None
Necator New World hookworm disease None
Strongyloides stercoralis Strongyloidiasis None
Trichinella spiralis Trichinosis None
Trichuris trichiura Whipworm infection None
Brugia malayi Filariasis Peptidylprolyl isomerase [45], [46]
Dracunculus medinensis Guinea worm disease None
Loa loa Loiasis None
Onchocerca volvulus River blindness None
Toxocara canis Visceral larva migrans None
Wuchereria bancrofti Lymphatic filariasis (elephantiasis) None
References: [1] Leonard et al. (1997); [2] Liu et al. (1998); [3] Phillips et al. (1999); [4] Knighton et al. (1994); [5] Kim et al. (1995); [6] Schlagenhauf et al.
(1998); [7] Shi, Schramm & Almo (1999); [8] Rigden et al. (1999); [9] Williams et al. (1999); [10] Kim et al. (1998); [11] Read et al. (1999); [12] Chitarra et al.
(1999); [13] Silva et al. (1996); [14] Shi, Li et al. (1999); [15] Velanker et al. (1997); [16] Champness et al. (1994); [17] Schumacher et al. (1996); [18]
Schumacher et al. (1998); [19] Chudzik et al. (2000); [20] Vellieux et al. (1993); [21] Phillips et al. (1998); [22] Bernstein et al. (1998); [23] Wierenga et al.
(1987); [24] Freymann et al. (1990); [25] McGrath et al. (1995); [26] Souza et al. (1998); [27] Focia et al. (1998); [28] Maldonado et al. (1998); [29] Lantwin et al.
(1994); [30] Blankenfeldt et al. (1999); [31] Yang & Moffat (1995); [32] Whitlow et al. (1997); [33] Weston et al. (1998); [34] Cleasby et al. (1996); [35] Cutfield
et al. (1995); [36] Kohno et al. (1996); [37] Suguna et al. (1987); [38] Kurihara et al. (1992); [39] Rossjohn, Feil, Wilce et al. (1997); [40] McTigue et al. (1995);
[41] Mulichak et al. (1998); [42] Yang et al. (1995); [43] Bullock et al. (1996); [44] Huang et al. (1994); [45] Mikol et al. (1998); [46] Taylor et al. (1998).
threatens the effective treatment of bacterial infections of

essentially every person on this planet. This implies that we will
constantly have to be aware of the potential occurrence of mono-
Table 1.3.4.4. Mechanisms of resistance and also multi-drug resistance, which has profound consequences
for drug-design strategies for essentially all infectious diseases. It
Overexpress target protein requires the development of many different compounds attacking
Mutate target protein many different target proteins and nucleic acids in the infectious
Use other protein with same function agent. It appears to be crucial to use, from the very beginning,
Remove target altogether several new drugs in combination so that the chances of the
Overexpress detoxification enzyme
occurrence of resistance are minimal. Multi-drug regimens have
been spectacularly successful in the case of leprosy and HIV.
Mutate detoxification enzyme
Obviously, the development of vaccines is by far the better solution,
Create new detoxification enzyme but it is not always possible. Antigenic variation, see e.g. the
Mutate membrane porin protein influenza virus, requires global vigilance and constant re-engineer-
Remove or underexpress membrane porin protein ing of certain vaccines every year. Moreover, for higher organisms,
Overexpress efflux pumps and even for many bacterial species like Shigella (Levine &
Mutate efflux pumps Noriega, 1995), with over 50 serotypes per species, the develop-
Create/steal new efflux pumps ment of successful vaccines has, unfortunately, proved to be very
Improve DNA repair difficult indeed. For sleeping sickness, the development of a vaccine
Mutate prodrug conversion enzyme is generally considered to be impossible. It is most likely, therefore,
that world health will depend for centuries on a wealth of
20
therapeutic drugs, together with many other measures, in order to resistance in cancer. The resistance is caused by cellular pumps
keep the immense number of pathogenic organisms under control. that efficiently pump out the drugs, often leading to failed
chemotherapy (Borst, 1999). On the other hand, the structures of
1.3.4.3. Non-communicable diseases major oncogenic proteins such as p21 (DeVos et al., 1988; Pai et al.,
1989; Krengel et al., 1990; Scheffzek et al., 1997) and p53 (Cho et
Of this large and diverse category of human afflictions we have
al., 1994; Gorina & Pavletich, 1996) are of tremendous importance
already touched upon genetic disorders in Section 1.3.3 above.
for future structure-based design of anti-neoplastic agents.
Other major types of non-communicable diseases include cancer,
aging disorders, diabetes, arthritis, and cardiovascular and
neurological illnesses. The field of non-communicable diseases is 1.3.4.3.2. Diabetes
immense. Describing in any detail the current projects in, and
potential impact of, protein and nucleic acid crystallography on The hallmark characteristic of type I diabetes is a lack of insulin.
these diseases would need more space than this entire volume on A major therapeutic approach to this problem is insulin replacement
macromolecular crystallography. Hence, only a few selected therapy. Unfortunately, the insulin requirements of the body vary
examples out of the hundreds which could be described can be dramatically during the course of a day, with high concentrations
discussed here. Table 1.3.4.5 lists many examples of human protein needed at meal times and a basal level during the rest of the day.
structures elucidated without any claim as to completeness – it is Only monomeric insulin is active at the insulin receptor level, but
simply impossible to keep up with the fountain of structures being insulin has a natural tendency to form dimers and hexamers that
determined at present. Yet, such tables do provide, it is hoped, an dissociate upon dilution. Thanks to the three-dimensional insight
overview of what has been achieved and what needs to be done. obtained from dozens of insulin crystal structures, as wild-type
(Hodgkin, 1971), mutants (Whittingham et al., 1998) and in
1.3.4.3.1. Cancers complex with zinc ions and small molecules such as phenol
(Derewenda et al., 1989), it has been possible to fine-tune the
Over a hundred different cancers have been described and clearly kinetics of insulin dissociation. The resulting availability of a
the underlying defect, loss of control of cell division, can be the variety of insulin preparations with rapid or prolonged action
result of many different shortcomings in different cells. The profiles has improved the quality of life of millions of people
research in this area proceeds at a feverish pace, yet the (Brange, 1997).
development, discovery and design of effective but safe anti-cancer
agents are unbelievably difficult challenges. The modifications
needed to turn a normal cell into a malignant one are very small, 1.3.4.3.3. Blindness
hence the chance of arriving at ‘true’ anti-cancer drugs that exploit
such small differences between normal and abnormal cells is The main causes of blindness worldwide are cataract, trachoma,
exceedingly small. Nevertheless, such selective anti-cancer agents glaucoma and onchocerciasis (Thylefors et al., 1995). Trachoma
would leave normal cells essentially unaffected and are therefore and onchocerciasis are parasitic diseases that destroy the
the holy grail of cancer therapy. Few if any such compounds have architecture of the eye; they were discussed in Section 1.3.4.1.
been found so far, but cancer therapy is benefiting from a gradual The other two are discussed here. During cataract development, the
increase in the number of useful compounds. Many have serious lens of the eye becomes non-transparent as a result of aggregation of
side effects, weaken the immune system and are barely tolerated by crystallins, preventing image formation. Crystal structures of
patients. However, they rescue large numbers of patients and hence several mammalian beta- and gamma-crystallins are known, but
it is of interest that many targets of these compounds, proteins and no human ones yet. In glaucoma, the optic nerve is destroyed by
DNA molecules, have been structurally elucidated by crystal- high intra-ocular pressure. One way to lower the pressure is to
lographic methods – often in complex with the cancer drug. The inhibit carbonic anhydrase II, a pivotal enzyme in maintaining the
mode of action of many anti-cancer compounds is well understood, intra-ocular pressure. On the basis of the carbonic anhydrase crystal
e.g. methotrexate targeting dihydrofolate reductase, and fluoro- structure, researchers at Merck Research Laboratories were able to
uracil targeting thymidilate synthase. These are specific enzyme guide the optimization of an S-thienothiopyran-2-sulfonamide lead
inhibitors acting along principles well known in other areas of into a marketed drug for glaucoma: dorzolamide (Baldwin et al.,
medicine. Several anti-cancer drugs display unusual modes of 1989).
action, such as:
(a) the DNA intercalators daunomycin (Wang et al., 1987) and
1.3.4.3.4. Cardiovascular disorders
adriamycin (Zhang et al., 1993);
(b) cisplatin, which forms DNA adducts (Giulian et al., 1996); Thrombosis is a major cause of morbidity and mortality,
(c) taxol, which not only binds to tubulin but also to bcl-2, especially in the industrial world. Hence, major effort is expended
thereby blocking the machinery of cancer cells in two entirely by pharmaceutical industries in the development of new classes of
different ways (Amos & Lowe, 1999); anti-coagulants with fewer side effects than available drugs, such as
(d ) camptothecin analogues, such as irinotecan and topotecan, heparins and coumarins. Because blood coagulation is the result of
which have the unusual property of prolonging the lifetime of a an amplification cascade of enzymatic reactions, many potential
covalent topoisomerase–DNA complex, generating major road targets are available. At present most of the effort is directed
blocks on the DNA highway and causing DNA breakage and cell towards thrombin (Weber & Czarniecki, 1997) and factor Xa
death; (Ripka, 1997), responsible for the penultimate step and the step
(e) certain compounds function as minor-groove binders, e.g. immediately preceding it in the cascade, respectively. Thrombin is
netropsin and distamycin (Kopka et al., 1985); especially fascinating owing to the presence of at least three
( f ) completely new drugs which were developed based on the subsites: a primary specificity pocket with the catalytic serine-
structures of matrix metalloproteinases, purine nucleotide phos- protease machinery, an exosite for recognizing extended fibrinogen
phorylase and glycinamide ribonucleotide formyltransferase and and an additional pocket for binding heparin. This knowledge has
which are in clinical trials (Jackson, 1997). led to the design of bivalent inhibitors which occupy two sites with
Meanwhile, it is sad that crystallography has not yet made any ultra-high affinity and exquisite specificity. Several of these agents
contribution to the molecular understanding of multi-drug are in clinical trials (Pineo & Hull, 1999).
21
1. INTRODUCTION
Table 1.3.4.5. Important human protein structures in drug design
Proteins from other species that might have been studied as substitutes for human ones were left out because of space limitations. We apologize to the researchers
affected.
Pharmacological category Protein Reference

Synaptic and neuroeffector junctional function None
Central nervous system function None
Inflammation Fibroblast collagenase (MMP-1) (also important in cancer) [1], [2], [3], [4]
Gelatinase [5], [6]
Stromelysin-1 (MMP-3) (also important in cancer) [7], [8], [9], [10], [11]
Matrilysin (MMP-7) (also important in cancer) [12]
Neutrophil collagenase (MMP-8) (also important in cancer) [13], [14], [15], [16]
Collagenase-3 (MMP-13) [17]
Human neutrophil elastase (also important for cystic fibrosis) [18], [19], [20]
Interleukin-1 beta converting enzyme (ICE) [21], [22]
p38 MAP kinase [23], [24]
Phospholipase A2 [25], [26], [27]
Renal and cardiovascular function Renin [28]
Gastrointestinal function None
Cancer 17-Beta-hydroxysteroid dehydrogenase [29], [30]
BRCT domain (BRCA1 C-terminus) [31]
Bcr-Abl kinase [32]
Cathepsin B [33]
Cathepsin D [34], [35]
CDK2 [36]
CDK6 [37]
DHFR [38], [39]
Acidic fibroblast growth factor (FGF) [40]
FGF receptor tyrosine kinase domain [41]
Glycinamide ribonucleotide formyl transferase [42]
Interferon-beta [43]
MMPs: see Inflammation
p53 [44], [45]
p60 Src [46]
Purine nucleoside phosphorylase [47]
ras p21 [48], [49], [50], [51]
Serine hydroxymethyltransferase [52]
S-Adenosylmethionine decarboxylase [53]
Thymidylate synthase [54]
Topoisomerase I [55], [56]
Tumour necrosis factor [57]
Interleukin 1-alpha [58]
Interleukin 1-beta [59]
Interleukin 1-beta receptor [60], [61]
Interleukin 8 [62]
Immunomodulation Calcineurin [63]
Cathepsin S [64]
Cyclophilin [65], [66], [67]
Immunophilin FKBP12 [68], [69], [70]
Inosine monophosphate dehydrogenase [71]
Interferon-gamma [72], [73]
Lymphocyte-specific kinase Lck [74]
PNP [47]
Syk kinase [75]
Tumour necrosis factor [57]
ZAP Tyr kinase [76]
Interleukin 2 [77]
Interleukin 5 [78]
Haematopoiesis Erythropoietin receptor [79], [80]
22
Table 1.3.4.5. Important human protein structures in drug design (cont.)
Pharmacological category Protein Reference

Coagulation AT III [81], [82], [83], [84]
Factor III [85], [86]
Factor VII [87]
Factor IX [88]
Factor X [89]
Factor XIII [90]
Factor XIV [91]
Fibrinogen: fragment [92], [93]
Plasminogen activator inhibitor (PAI) [94], [95], [96]
Thrombin [97], [98], [99]
tPA [100]
Urokinase-type plasminogen activator [101]
von Willebrand factor [102], [103], [104]
Hormones and hormone receptors Insulin [105]
Insulin receptor [106], [107]
Human growth hormone + receptor [108]
Oestrogen receptor [109], [110]
Progesterone receptor [111]
Prolactin receptor [112]
Ocular function Carbonic anhydrase [113]
Genetic diseases See Table 1.3.3.1
Drug binding Human serum albumin [114], [115]
Drug metabolism Glutathione S-transferase A-1 [116], [117]
Glutathione S-transferase A4-4 [118]
Glutathione S-transferase Mu-1 [119]
Glutathione S-transferase Mu-2 [120]
Neurodegeneration Aldose reductase [121]
JNK3 [122]
Osteoporosis Cathepsin K [123], [64]
Src SH2 [126]
Various Interferon-alpha 2b [124]
Bcl-xL [125]
References: [1] Borkakoti et al. (1994); [2] Lovejoy, Cleasby et al. (1994); [3] Lovejoy, Hassell et al. (1994); [4] Spurlino et al. (1994); [5] Libson et al. (1995); [6]
Gohlke et al. (1996); [7] Becker et al. (1995); [8] Dhanaraj et al. (1996); [9] Esser et al. (1997); [10] Gomis-Ruth et al. (1997); [11] Finzel et al. (1998); [12]
Browner et al. (1995); [13] Bode et al. (1994); [14] Reinemer et al. (1994); [15] Stams et al. (1994); [16] Betz et al. (1997); [17] Gomis-Ruth et al. (1996); [18]
Bode et al. (1986); [19] Wei et al. (1988); [20] Navia, McKeever et al. (1989); [21] Walker et al. (1994); [22] Rano et al. (1997); [23] Wilson et al. (1996); [24]
Tong et al. (1997); [25] Scott et al. (1991); [26] Wery et al. (1991); [27] Kitadokoro et al. (1998); [28] Sielecki et al. (1989); [29] Ghosh et al. (1995); [30] Breton
et al. (1996); [31] Zhang et al. (1998); [32] Nam et al. (1996); [33] Musil et al. (1991); [34] Baldwin et al. (1993); [35] Metcalf & Fusek (1993); [36] De Bondt et
al. (1993); [37] Russo et al. (1998); [38] Oefner et al. (1988); [39] Davies et al. (1990); [40] Blaber et al. (1996); [41] McTigue et al. (1999); [42] Varney et al.
(1997); [43] Karpusas et al. (1997); [44] Cho et al. (1994); [45] Gorina & Pavletich (1996); [46] Xu et al. (1997); [47] Ealick et al. (1990); [48] DeVos et al.
(1988); [49] Pai et al. (1989); [50] Krengel et al. (1990); [51] Scheffzek et al. (1997); [52] Renwick et al. (1998); [53] Ekstrom et al. (1999); [54] Schiffer et al.
(1995); [55] Redinbo et al. (1998); [56] Stewart et al. (1998); [57] Banner et al. (1993); [58] Graves et al. (1990); [59] Priestle et al. (1988); [60] Schreuder et al.
(1997); [61] Vigers et al. (1997); [62] Baldwin et al. (1991); [63] Kissinger et al. (1995); [64] McGrath et al. (1998); [65] Kallen et al. (1991); [66] Ke et al. (1991);
[67] Pfuegl et al. (1993); [68] Van Duyne, Standaert, Karplus et al. (1991); [69] Van Duyne, Standaert, Schreiber & Clardy (1991); [70] Van Duyne et al. (1993);
[71] Colby et al. (1999); [72] Ealick et al. (1991); [73] Walter et al. (1995); [74] Zhu et al. (1999); [75] Futterer et al. (1998); [76] Meng et al. (1999); [77]
Brandhuber et al. (1987); [78] Milburn et al. (1993); [79] Livnah et al. (1996); [80] Livnah et al. (1998); [81] Carrell et al. (1994); [82] Schreuder et al. (1994);
[83] Skinner et al. (1997); [84] Skinner et al. (1998); [85] Muller et al. (1994); [86] Muller et al. (1996); [87] Banner et al. (1996); [88] Rao et al. (1995); [89]
Padmanabhan et al. (1993); [90] Yee et al. (1994); [91] Mather et al. (1996); [92] Pratt et al. (1997); [93] Spraggon et al. (1997); [94] Mottonen et al. (1992); [95]
Aertgeerts et al. (1995); [96] Xue et al. (1998); [97] Bode et al. (1989); [98] Rydel et al. (1990); [99] Rydel et al. (1994); [100] Laba et al. (1996); [101] Spraggon
et al. (1995); [102] Bienkowska et al. (1997); [103] Huizinga et al. (1997); [104] Emsley et al. (1998); [105] Ciszak & Smith (1994); [106] Hubbard et al. (1994);
[107] Hubbard (1997); [108] DeVos et al. (1992); [109] Schwabe et al. (1993); [110] Brzozowski et al. (1997); [111] Williams & Sigler (1998); [112] Somers et al.
(1994); [113] Kannan et al. (1975); [114] He & Carter (1992); [115] Curry et al. (1998); [116] Sinning et al. (1993); [117] Cameron et al. (1995); [118] Bruns et al.
(1999); [119] Tskovsky et al. (1999); [120] Raghunathan et al. (1994); [121] Wilson et al. (1992); [122] Xie et al. (1998); [123] Thompson et al. (1997); [124]
Radhakrishnan et al. (1996); [125] Muchmore et al. (1996); [126] Waksman et al. (1993).
23
1. INTRODUCTION
1.3.4.3.5. Neurological disorders cytochrome P-450, the most important class of xenobiotic
metabolizing enzymes, has been reported (Williams et al., 2000).
Even a quick glance at Table 1.3.4.5 shows that crystallography
Of the conjugation enzymes, only glutathione S-transferases (EC
contributes to new therapeutics for numerous human afflictions and
2.5.1.18) have been characterized structurally: A1 (Sinning et al.,
diseases. Yet there are major gaps in our understanding of protein
1993), A4-4 (Bruns et al., 1999), MU-1 (Patskovsky et al., 1999),
functions, in particular of those involved in development and in
MU-2 (Raghunathan et al., 1994), P (Reinemer et al., 1992) and
neurological functions. These proteins are the target of many drugs
THETA-2 (Rossjohn, McKinstry et al., 1998). Tens of structures
obtained by classical pre-crystal-structure methods. These proven
await elucidation in this area (Testa, 1994).
drug targets are very often membrane proteins involved in neuronal
functions, and the diseases concerned are some of the most
1.3.4.5. Drug manufacturing and crystallography
prevalent in mankind. A non-exhaustive list includes cerebro-
vascular disease (strokes), Parkinson’s, epilepsy, schizophrenia, The development of drugs is a major undertaking and one of the
bipolar disease and depression. hallmarks of modern societies. However, once a safe and effective
Some of these diseases are heart-breaking afflictions, where therapeutic agent has been fully tested and approved, manufacturing
parents have to accept the suicidal tendencies of their children, the compound on a large scale is often the next major challenge.
often with fatal outcomes; where partners have to endure the Truly massive quantities of penicillin and cephalosporin are
tremendous mood swings of their bipolar spouses and have to produced worldwide, ranging from 2000 to 7000 tons annually
accept extreme excesses in behaviour; where a happy evening of (Conlon et al., 1995). In the production of semi-synthetic
life is turned into the gradual and sad demise of human intellect due penicillins, the enzyme penicillin acylase plays a very significant
to the progression of Alzheimer’s, or to the loss of motor functions role. This enzyme catalyses the hydrolysis of penicillin into
due to Parkinson’s, or into the tragic stare of a victim of deep 6-aminopenicillanic acid. Its crystal structure has been elucidated
depression. Human nature, in all its shortcomings, has the tendency (Duggleby et al., 1995) and may now be used for protein-
to try to help such tragic victims, but drugs for neurological engineering studies to improve its properties for the biotechnology
disorders are rare, drug regimens are difficult to optimize and the industry. The production of cephalosporins could benefit in a
commitment to follow a drug regimen – often for years, and often similar way from knowing the structure of cephalosporin acylase
with major side effects – is a next to impossible task in many cases. (CA), since the properties of this enzyme are not optimal for use in
New, better drugs are urgently needed and hence the structure production plants. Therefore, the crystal structure determination of
determinations of the ‘molecules of the brain’ are major scientific as CA could provide a basis for improving the substrate specificity of
well as medical challenges of the next decades. Such molecules will CA by subsequent protein-engineering techniques. Fortunately, a
shed light on some of the deepest mysteries of humanity, including first CA structure has been solved recently (Kim et al., 2000), with
memory, cognition, desire, sleep etc. At the same time, such many other structures expected to be solved essentially simulta-
structures will provide opportunities for treating those suffering neously. Clearly, crystallography can be not only a major player in
from neurodegenerative diseases due to age, genetic disposition, the design and optimization of therapeutic drugs, but also in their
allergies, infections, traumas and combinations thereof. Such ‘CNS manufacture.
protein structures’ are one of the major challenges of biomacro-
molecular crystallography in the 21st century.
1.3.4.4. Drug metabolism and crystallography 1.3.5. Vaccines, immunology and crystallography
As soon as a drug enters the body, an elaborate machinery comes Vaccines are probably the most effective way of preventing disease.
into action to eliminate this foreign and potentially harmful An impressive number of vaccines have been developed and many
molecule as quickly as possible. Two steps are usually distinguished more are under development (National Institute of Allergy and
in this process: phase I metabolism, in which the drug is Infectious Diseases, 1998). Smallpox has been eradicated thanks to
functionalized, and phase II metabolism, in which further a vaccine, and polio is being targeted for eradication in a worldwide
conjugation with endogenous hydrophilic molecules takes place, effort, again using vaccination strategies. To the best of our
so that excretion via the kidneys can occur. Whereas this knowledge, crystal structures of viruses, viral capsids or viral
‘detoxification’ process is essential for survival, it often renders proteins have not been used in developing the currently available
promising inhibitors useless as drug candidates. Hence, structural vaccines. However, there are projects underway that may change
knowledge of the proteins involved in metabolism could have a this.
significant impact on the drug development process. For instance, the crystal structure of rhinovirus has resulted in the
Thus far, only the structures of a few proteins crucial for drug development of compounds that have potential as antiviral agents,
distribution and metabolism have been elucidated. Human serum since they stabilize the viral capsid and block, or at least delay, the
albumin binds hundreds of different drugs with micromolar uncoating step in viral cell entry (Fox et al., 1986). These rhinovirus
dissociation constants, thereby altering drug levels in the blood capsid-stabilizing compounds are, in a different project, being used
dramatically. The structure of this important carrier molecule has to stabilize poliovirus particles against heat-induced denaturation in
been solved in complex with several drug molecules and should one vaccines (Grant et al., 1994). This approach may be applicable to
day allow the prediction of the affinity of new chemical entities for other cases, although it has not yet resulted in commercially
this carrier protein, and thereby deepen our understanding of the available vaccine-plus-stabilizer cocktails. However, it is fascinat-
serum concentrations of new candidate drugs (Carter & Ho, 1994; ing to see how a drug-design project may be able to assist vaccine
Curry et al., 1998; Sugio et al., 1999). Human oxidoreductases and development in a rather unexpected manner.
hydrolases of importance in drug metabolism with known structure Three-dimensional structural information about viruses is also
are: alcohol dehydrogenase (EC 1.1.1.1) (Hurley et al., 1991), being used to aid in the development of vaccines. Knowledge of the
aldose reductase (EC 1.1.1.21) (Wilson et al., 1992), glutathione architecture of and biological functions of coat proteins has been
reductase (NADPH) (EC 1.6.4.2) (Thieme et al., 1981), catalase used to select loops at viral surfaces that can be replaced with
(EC 1.11.1.6) (Ko et al., 2000), myeloperoxidase (EC 1.11.1.7) antigenic loops from other pathogens for vaccine-engineering
(Choi et al., 1998) and beta-glucuronidase (EC 3.2.1.31) (Jain et al., purposes (e.g. Burke et al., 1988; Kohara et al., 1988; Martin et
1996). Recently, the first crystal structure of a mammalian al., 1988; Murray et al., 1998; Arnold et al., 1994; Resnick et al.,
24
1995; Smith et al., 1998; Arnold & Arnold, 1999; Zhang, Geisler et cruelly debilitating diseases such as rheumatoid arthritis and type I
al., 1999). The design of human rhinovirus (HRV) and poliovirus diabetes.
chimeras has been aided by knowing the atomic structure of the
viruses (Hogle et al., 1985; Rossmann et al., 1985; Arnold &
Rossmann, 1988; Arnold & Rossmann, 1990) and detailed features 1.3.6. Outlook and dreams
of the neutralizing immunogenic sites on the virion surfaces (Sherry
At the beginning of the 1990s, Max Perutz inspired many
& Rueckert, 1985; Sherry et al., 1986). In this way, one can imagine
researchers with a passion for structure and a heart for the suffering
that in cases where the atomic structures of antigenic loops in
of mankind with a fascinating book entitled Protein Structure –
‘donor’ immunogens are known as well as the structure of the
New Approaches to Disease and Therapy (Perutz, 1992). The
‘recipient’ loop in the virus capsid protein, optimal loop
explosion of medicinal macromolecular crystallography since then
transplantation might become possible. It is not yet known how to
has been truly remarkable. What should we expect for the next
engineer precisely the desired three-dimensional structures and
decades?
properties into macromolecules. However, libraries of macromole-
In the realm of safe predictions we can expect the following:
cules or viruses constructed using combinatorial mutagenesis can be
(a) High-throughput macromolecular crystallography due to the
searched to increase the likelihood of including structures with
developments outlined in Section 1.3.1, leading to the new field of
desired architecture and properties such as immunogenicity. With
‘structural genomics’.
appropriate selection methods, the rare constructs with desired
(b) Crystallography of very large complexes. While it is now
properties can be identified and ‘fished out’. Research of this type
clear that an atomic structure of a complex of 58 proteins and three
has yielded some potently immunogenic presentations of sequences
RNA molecules, the ribosome, is around the corner, crystal-
transplanted on the surface of HRV (reviewed in Arnold & Arnold,
lographers will widen their horizons and start dreaming of
1999). For reasons not quite fully understood, presenting multiple
structures like the nuclear pore complex, which has a molecular
copies of antigens to the immune system leads to an enhanced
weight of over 100 000 000 Da.
immune response (Malik & Perham, 1997). It is conceivable that,
(c) A steady flow of membrane protein structures. Whereas Max
eventually, it might even be possible for conformational epitopes
Perutz could only list five structures in his book of 1992, there are
consisting of multiple ‘donor’ loops to be grafted onto ‘recipient
now over 40 PDB entries for membrane proteins. Most of them are
capsids’ while maintaining the integrity of the original structure.
transmembrane proteins: bacteriorhodopsin, photoreaction centres,
Certainly, such feats are difficult to achieve with present-day
light-harvesting complexes, cytochrome bc1 complexes, cyto-
protein-engineering skills, but recent successes in protein design
chrome c oxidases, photosystem I, porins, ion channels and
offer hope that this will be feasible in the not too distant future
bacterial toxins such as haemolysin and LukF. Others are
(Gordon et al., 1999).
monotopic membrane proteins such as squalene synthase and the
Immense efforts have been made by numerous crystallographers
cyclooxygenases. Clearly, membrane protein crystallography is
to unravel the structures of molecules involved in the unbelievably
gaining momentum at present and may open the door to atomic
complex, powerful and fascinating immune system. Many of the
insight in neurotransmitter pharmacology in the next decade.
human proteins studied are listed in Table 1.3.4.5 with, as specific
What if we dream beyond the obvious? One day, medicinal
highlights, the structures of immunoglobulins (Poljak et al., 1973),
crystallography may contribute to:
major histocompatibility complex (MHC) molecules (Bjorkman et
(a) The design of submacromolecular agonists and antagonists of
al., 1987; Brown et al., 1993; Fremont et al., 1992; Bjorkman &
proteins and nucleic acids in a matter of a day by integrating rapid
Burmeister, 1994), T-cell receptors (TCR) and MHC:TCR
structure determinations, using only a few nanograms of protein,
complexes (Garboczi et al., 1996; Garcia et al., 1996), an array of
with the power of combinatorial and, in particular, computational
cytokines and chemokines, and immune cell-specific kinases such
chemistry.
as lck (Zhu et al., 1999). This knowledge is being converted into
(b) ‘Structural toxicology’ based on ‘human structural geno-
practical applications, for instance by humanising non-human
mics’. Once the hundreds of thousands of structures of human
antibodies with desirable properties (Reichmann et al., 1988) and
proteins and complexes with other proteins and nucleic acids have
by creating immunotoxins.
been determined, truly predictive toxicology may become possible.
The interactions between chemokines and receptors, and the
This will not only speed up the drug-development process, but may
complicated signalling pathways within each immune cell, make it
substantially reduce the suffering of animals in preclinical tests.
next to impossible to predict the effect of small compounds
(c) The creation of completely new classes of drugs to treat
interfering with a specific protein–protein interaction in the immune
addiction, organ regeneration, aging, memory enhancement etc.
system (Deller & Jones, 2000). However, great encouragement has
One day, crystallography will have revealed the structure of
been obtained from the discovery of the remarkable manner by
hundreds of thousands of proteins and nucleic acids from human
which the immunosuppressor FK506 functions: this small molecule
and pathogen, and their complexes with each other and with natural
brings two proteins, FKB12 and calcineurin, together, thereby
and designed low-molecular-weight ligands. This will form an
preventing T-cell activation by calcineurin. The structure of this
extraordinarily precious database of knowledge for furthering the
remarkable ternary complex is known (Kissinger et al., 1995). Such
health of humans. Hence, in the course of the 21st century,
discoveries of unusual modes of action of therapeutic compounds
crystallography is likely to become a major driving force for
are the foundation for new concepts such as ‘chemical dimerizers’
improving health care and disease prevention, and will find a well
to activate signalling events in cells such as apoptosis (Clackson et
deserved place in future books describing progress in medicine,
al., 1998).
sometimes called ‘The Greatest Benefit to Mankind’ (Porter, 1999).
In spite of the gargantuan task ahead aimed at unravelling the
cell-to-cell communication in immune action, it is unavoidable that
the next decades will bring us unprecedented insight into the many
Acknowledgements
carefully controlled processes of the immune system. In turn, it is
expected that this will lead to new therapeutics for manipulating a We wish to thank Heidi Singer for terrific support in preparing the
truly wonderful defence system in order to assist vaccines, to manuscript, and Drs Alvin Kwiram, Michael Gelb, Seymour
decrease graft rejection processes in organ transplants and to control Klebanov, Wes Van Voorhis, Fred Buckner, Youngsoo Kim and
auto-immune diseases that are likely to be playing a major role in Rein Zwierstra for valuable comments.
25
references
1.4. Perspectives for the future

BY E. ARNOLD AND M. G. ROSSMANN
1.4.1. Gazing into the crystal ball (E. ARNOLD) damage by physical and chemical agents. Now, materials science
includes key foci in development of new biomaterials and in the
We live in an era when there are many wonderful opportunities burgeoning field of nanotechnology. The acrobatics of new smart
for reaching new vistas of human experience. Some of the dreams materials could include computation at speeds that may be much
that we hold may be achieved through scientific progress. Among faster than can be accomplished with silicon-based materials.
the things we have learned in scientific research is to expect the
unexpected – the crystal ball holds many surprises for us in
the future. Alchemists of ancient times laboured to turn ordinary 1.4.1.2. How will crystallography change in the future?
materials into precious metals. These days, scientists can create
substances far more precious than gold by discovering new Potential future advances in the fields of crystallography,
medicines and materials for high-technology industries. Crystal- structural chemistry and biology are tantalizing. Successful imaging
lography has played an important role in helping to advance science of single specimens and single molecules at high resolution may
and the human endeavour in the twentieth century. I expect to see eventually be achievable. Owing to the limitations of current
great contributions from crystallography also in the twenty-first physical theories and experimental possibilities, large numbers of
century. molecules have generally been required for detailed investigations
Science of the twentieth century has yielded a great deal of of molecular phenomena. Given the complexity of large biological
insight into the workings of the natural world. Systematic advances molecules, only techniques such as X-ray crystallography and NMR
are permitting dissection of the molecular anatomy of living have been suitable for describing the detailed atomic structures of
systems. This has propelled us into a world where these insights these systems. It may eventually be possible to use X-ray
can be brought to bear on problems of design. The impact in such microscopy to obtain detailed images of even single specimens.
fields as health and medicine, materials science, and microelec- Merging of information from multiple specimens as is currently
tronics will be continually greater. done in electron microscopy may be very powerful in X-ray
microscopy as well.
X-ray sources will continue to evolve. High-intensity synchro-
1.4.1.1. What can we expect to see in the future of science
tron sources have allowed the development of dramatically faster
and technology in general?
and higher-quality diffraction data measurements. Complete multi-
Just as few were successful in predicting the ubiquitous impact of wavelength X-ray diffraction data sets have been measured from
the internet, it is difficult to predict which specific technologies will frozen protein crystals containing selenomethionine (SeMet) in less
accomplish the transition into the culture of the future. It is possible than one hour, leading to nearly automatic structure solution by the
to envision instantaneous telecommunication and videoconferen- multiwavelength anomalous diffraction (MAD) technique. At
cing with colleagues and friends throughout the world – anytime, present, such synchrotron facilities are enormous national or
anywhere – using small, portable devices. Access to computer- multinational facilities that sap the electric power of an entire
based information via media such as the internet will become region. Perhaps portable X-ray sources will be developed that can
continually more facile and powerful. This will permit access to the be used to create synchrotron-like intensity in the laboratory. If such
storehouse of human knowledge in unprecedented ways, catalysing sources could have a tunable energy or wavelength, then
more rapid development of new ideas. experiments such as MAD would be routinely accessible within
Experimental tests of new ideas will continue to play a crucial the laboratory. Better time resolution of molecular motion and of
role in the guidance of scientific knowledge and reasoning. chemical reactions will be achieved with higher-intensity sources.
However, more powerful computing resources may change Sample preparation for macromolecular structural studies has
paradigms in which ever more powerful simulation techniques undergone a complete revolution thanks to the advent of
can bootstrap from primitive ideas to full-blown theories. I still recombinant DNA methods. Early macromolecular studies were
expect that experiment will be necessary for the foreseeable future, limited to materials present in large abundance. By the late 1970s
since nearly every well designed experiment yields unexpected and early 1980s, molecular biology made it possible to obtain
results, often at a number of levels. desired gene products in large amounts, and new methods of
In the realm of biology, greater understanding of the structure chemical synthesis permitted production of large quantities of
and mechanism of living processes will permit unprecedented defined oligonucleotides. Initial drafts of the entire human genome
advances in health and medicine. Even those scientists most have been mapped and sequenced, allowing even broader access to
sceptical of molecular-design possibilities would be likely to genes for study. The combination of structural genomics and
admit that revolutionary advances have been achieved. In the area already ongoing studies will lead to knowing the structure of the
of drug design, for example, structure-based approaches have entire human proteome in a finite amount of time. Many materials
yielded some of the most important new molecules currently being are still challenging to produce in quantities sufficient for structural
introduced worldwide for the treatment of human diseases ranging studies. Engineering methods (site-directed mutagenesis, combina-
from AIDS and influenza to cancer and heart disease. This is a torial mutagenesis and directed evolution techniques) have
relatively young and very rapidly changing area, and it is reasonable permitted additional sampling of molecular diversity, and we can
to expect that we have witnessed only the tip of the iceberg. Dream expect that even more powerful methods will be developed.
drugs to control growth and form, aging, intelligence, and other Engineering of solubility and crystallizability will help make
physiologically linked aspects of health and well-being may be more problems tractable for study. Perhaps, as suggested before,
developed in our lifetime. As greater understanding of the structural techniques for visualization of single molecules may become
basis of immunogenicity emerges, we should also expect to benefit adequate for in situ visualization of molecular interactions in living
from structure-based approaches to vaccine design. cells and organisms. However, traditional considerations of amount,
Other areas where molecular design will play revolutionary roles purity, specific activity etc. will remain important, as will hard work
include the broad field of material sciences. Traditionally, and good luck.
‘materials science’ referred to the development of materials with The phase problem has continued to be a stumbling block for
desired physical properties – strength, flexibility, and resistance to structure determination. Experimental methods, including isomor-
26

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International Tables for Crystallography

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International Tables for Crystallography Mathematical

International Tables for Crystallography Space Group

Macromolecular Crystallography Protocols Volume 1

Energetics of Biological Macromolecules 1st Edition Jo

NMR Crystallography 1st Edition Robin K. Harris

Single particle Cryo EM of Biological Macromolecules

Macromolecular Crystallography MIE 1st Edition Alfred

Fundamentals of X Ray Crystallography 2nd Edition

Volume A: Space-Group Symmetry

Volume B: Reciprocal Space

Volume C: Mathematical, Physical and Chemical Tables

Volume F: Crystallography of Biological Macromolecules

Volume D: Physical Properties of Crystals

Volume E: Subperiodic Groups

Volume A1: Symmetry Relations between Space Groups

Published by Kluwer Academic Publishers,

Technical Editor: N. J. Ashcroft

1.1. Overview (E. Arnold and M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1

1.2. Historical background (M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 4

1.3. Macromolecular crystallography and medicine (W. G. J. Hol and C. L. M. J. Verlinde) .. .. .. .. .. .. .. .. .. .. .. .. 10

1.4. Perspectives for the future .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 26

PART 2. BASIC CRYSTALLOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 45

2.1. Introduction to basic crystallography (J. Drenth) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 45

PART 3. TECHNIQUES OF MOLECULAR BIOLOGY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 65

4.1. General methods (R. GiegeÂ and A. McPherson) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 81

4.2. Crystallization of membrane proteins (H. Michel) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 94

PART 5. CRYSTAL PROPERTIES AND HANDLING .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 111

5.2. Crystal-density measurements (E. M. Westbrook) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 117

PART 6. RADIATION SOURCES AND OPTICS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 125

6.1. X-ray sources (U. W. Arndt) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 125

6.2. Neutron sources (B. P. Schoenborn and R. Knott) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 133

PART 7. X-RAY DETECTORS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 143

7.2. CCD detectors (M. W. Tate, E. F. Eikenberry and S. M. Gruner) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 148

PART 8. SYNCHROTRON CRYSTALLOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 155

8.2. Laue crystallography: time-resolved studies (K. Moffat) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 167

PART 9. MONOCHROMATIC DATA COLLECTION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 177

PART 10. CRYOCRYSTALLOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 197

10.1. Introduction to cryocrystallography (H. Hope) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 197

10.2. Cryocrystallography techniques and devices (D. W. Rodgers) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 202

PART 11. DATA PROCESSING .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 209

11.1. Automatic indexing of oscillation images (M. G. Rossmann) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 209

11.2. Integration of macromolecular diffraction data (A. G. W. Leslie) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 212

11.4. DENZO and SCALEPACK (Z. Otwinowski and W. Minor) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 226

PART 12. ISOMORPHOUS REPLACEMENT .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 247

12.2. Locating heavy-atom sites (M. T. Stubbs and R. Huber) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 256

PART 13. MOLECULAR REPLACEMENT .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263

13.1. Noncrystallographic symmetry (D. M. Blow) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 263

13.2. Rotation functions (J. Navaza) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 269

13.3. Translation functions (L. Tong) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 275

PART 14. ANOMALOUS DISPERSION .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 293

14.2. MAD and MIR .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 299

PART 15. DENSITY MODIFICATION AND PHASE COMBINATION .. .. .. .. .. .. .. .. .. .. .. .. .. 311

PART 16. DIRECT METHODS .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 333

16.2. The maximum-entropy method (G. Bricogne) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 346