Structural Characterization of Proteins and Complexes Using Small-Angle X-Ray Solution Scattering

Journal of Structural Biology 172 (2010) 128–141
Contents lists available at ScienceDirect
Journal of Structural Biology

journal homepage: www.elsevier.com/locate/yjsbi
Structural characterization of proteins and complexes using small-angle

X-ray solution scattering
Haydyn D.T. Mertens, Dmitri I. Svergun *
European Molecular Biology Laboratory-Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany
a r t i c l e i n f o a b s t r a c t
Article history: Small-angle scattering of X-rays (SAXS) is an established method for the low-resolution structural
Available online 15 June 2010 characterization of biological macromolecules in solution. The technique provides three-dimensional
low-resolution structures, using ab initio and rigid body modeling, and allow one to assess the oligomeric
Keywords: state of proteins and protein complexes. In addition, SAXS is a powerful tool for structure validation and
Small-angle scattering the quantitative analysis of flexible systems, and is highly complementary to the high resolution methods
Solution scattering of X-ray crystallography and NMR. At present, SAXS analysis methods have reached an advanced state,
Macromolecular structure
allowing for automated and rapid characterization of protein solutions in terms of low-resolution models,
Functional complexes
Ab initio methods
quaternary structure and oligomeric composition. In this communication, main approaches to the
Rigid body modeling characterization of proteins and protein complexes using SAXS are reviewed. The tools for the analysis
Flexible macromolecules of proteins in solution are presented, and the impact that these tools have made in modern structural
biology is discussed.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Production of good quality samples is a prerequisite for a suc-

cessful structural study by any method, and modern approaches
Small-angle scattering (SAS) of X-rays (SAXS) and neutrons to protein expression and purification used in structural biology
(SANS) is a powerful method for the analysis of biological macro- laboratories help to facilitate this. Like the high resolution methods
molecules in solution. Great progress has been made over the X-ray crystallography and nuclear magnetic resonance (NMR), SAS
years in applying this technique to extract structural information requires milligram amounts of highly pure, monodisperse protein
from non-crystalline samples in the fields of physics, materials that remains soluble at high concentration. However, while sample
science and biology (Feigin and Svergun, 1987). Over the last dec- requirements are similar for the three methods (noting that an
ade, major advances in instrumentation and computational meth- additional crystallization step is not required for the solution
ods have led to new and exciting developments in the application methods) a distinct advantage of SAXS is the speed of both data
of SAXS to structural biology. Active research is now being con- collection and sample characterization. On a modern synchrotron,
ducted by an increasing number of laboratories on advancing ab scattering data can be collected in seconds, allowing an almost
initio and rigid body modeling methods, the calculation of theo- immediate characterization of the sample and the sample quality
retical scattering curves from atomic models and the character- through the extraction of several overall parameters from the radi-
ization of quaternary structure and intrinsic flexibility. In ally averaged scattering pattern. SAXS can thus be used as a meth-
addition, advances in the automation of data collection and anal- od for the rapid screening of samples in various aqueous solvents/
ysis make high throughput applications of SAXS experiments additives, including e.g. identification and optimization of crystal-
tractable (Hura et al., 2009; Round et al., 2008). Such develop- lization conditions (Bonnete et al., 1999; Hamiaux et al., 2000).
ments have generated a renewed interest in the wider applica- In this review the discussion of SAS focusses on the elastic scat-
tions of the technique in the structural biology community. The tering of X-rays, SAXS, where dissolved macromolecules are exposed
present review is focused on the characterization of protein struc- to a collimated and (for synchrotrons) focussed X-ray beam and the
ture and complex formation, but the method is widely used for scattered intensity I is recorded by a detector as a function of the
other macromolecular structures, e.g. RNA (Doniach and Lipfert, scattering angle (Fig. 1A). For an in-depth review of the theory be-
2009; Rambo and Tainer, 2010). hind SAXS the reader is directed to text-books and recent reviews
(Feigin and Svergun, 1987; Koch et al., 2003; Putnam et al., 2007;
Svergun, 2007; Svergun and Koch, 2003; Tsuruta and Irving, 2008),
* Corresponding author. Fax: +49 40 89902 149. here some of the basic concepts will be presented with a focus on
E-mail address: [email protected] (D.I. Svergun). the characterization of proteins and protein complexes.
1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jsb.2010.06.012
H.D.T. Mertens, D.I. Svergun / Journal of Structural Biology 172 (2010) 128–141 129
Fig. 1. Schematic representation of a typical SAS experiment and radially averaged data. (A) Standard scheme of a SAS experiment. (B) X-ray scattering patterns from a
solution of BSA measured at X33 (DORIS, Hamburg) in 50 mM HEPES, pH 7.5, solvent scattering and the difference curve (containing the contribution from the protein alone,
scaled for the solute concentration, 5 mg/ml).
Scattering of X-rays by a solution of biomolecules is dependant SAXS is a technique that can probe structure on an extremely
on the number of biomolecules in the illuminated volume (i.e. to broad range of macromolecular sizes (Feigin and Svergun, 1987).
the solute concentration) and the excess scattering length density Small proteins and polypeptides in the range of 1–10 kDa, macro-
(often also called the contrast). For X-rays, the excess scattering molecular complexes and large viral particles up to several hun-
length density, Dq(r), comes from the difference in the electron dred MDa can all be measured with modern instrumentation
density of the solute and solvent which, for biomolecules in under near native conditions. It is often attractive to laboratory
aqueous solutions is very small. Consequently, synchrotron SAXS based researchers as the amount of material required for a com-
beamlines and laboratory sources must be optimized for the min- plete study is relatively low (typically 1–2 mg protein), and almost
imization of the contribution of background. any biologically relevant sample conditions can be used. The effect
Dilute aqueous solutions of proteins, nucleic acids or other mac- of changes to sample environment (pH, temperature, salt concen-
romolecules give rise to an isotropic scattering intensity, which de- tration and ligand/co-factor titration) can be easily measured
pends on the modulus of the momentum transfer s (s = 4psin(h)/k, and, moreover, at high-brilliance synchrotron beamlines time-re-
where 2h is the angle between the incident and scattered beam): solved experiments can be conducted (Lamb et al., 2008a,b; Pollack
and Doniach, 2009; West et al., 2008).
IðsÞ ¼ hIðsÞiX ¼ hAðsÞA ðsÞiX ð1Þ It should be noted here that the elastic scattering of neutrons
(SANS) is also widely used to characterize macromolecular solu-
where the scattering amplitude A(s) is a Fourier transformation of the
tions. Moreover, many approaches described below for SAXS are
excess scattering length density, and the scattering intensity is aver-
also applicable for SANS, where the excess scattering length den-
age over all orientations (X). Following subtraction of the solvent
sity (contrast) is due to the nuclear (and sometimes spin) scatter-
scattering, the background corrected intensity I(s) is proportional
ing length density instead of the electron density. In SANS, samples
to the scattering of a single particle averaged over all orientations.
highly absorbing to X-rays (e.g. solvents containing high salt) can
The scattering patterns generated from a dilute solution of mac-
be measured, and the samples will not suffer from radiation dam-
romolecules are typically presented as radially averaged one-
age. Most importantly, contrast variation by hydrogen/deuterium
dimensional curves (Fig. 1B). From these curves several overall
exchange can be used yielding precious additional information
important parameters can be directly obtained providing informa-
about the structure of macromolecular complexes. The disadvan-
tion about the size, oligomeric state and overall shape of the mol-
tages to SANS are that it usually requires more material than is re-
ecule. However, advances in computational methods have now
quired for SAXS, buffer subtraction is often difficult due to the high
made it possible to not only extract these simple parameters, but
incoherent hydrogen scattering and that the measurements cannot
to also determine reliable three-dimensional structures from scat-
be done on a laboratory source. Overall, SANS is a powerful com-
tering data. Low-resolution (1–2 nm) SAXS models can be deter-
plementary tool to SAXS (Ibel and Stuhrmann, 1975; Petoukhov
mined ab initio or through the refinement of available high-
and Svergun, 2006; Wall et al., 2000; Whitten and Trewhella,
resolution structures and/or homology models. While the former
2009).
analysis provides a low-resolution shape of the molecule in ques-
tion and often adds insight to the biological problem at hand, the
latter combination of SAXS and complementary data is a powerful 2. Overall SAXS parameters and rapid sample characterization
method for the determination of the organisation of macromolec-
ular complexes. In addition to structure determination SAXS is rou- Although sophisticated approaches have now been developed
tinely used for the validation of structural models, the quantitative for the determination of three-dimensional structure from scatter-
analysis of oligomeric state and the estimation of volume fractions ing data (see the following sections), several overall invariant
of components in mixtures/polydisperse systems. While SAXS has shape and weight parameters can be extracted directly from scat-
been readily employed for the analysis of flexible systems includ- tering curves enabling fast sample characterization. These param-
ing solutions of intrinsically unfolded proteins, methods were eters include: the molecular mass (MM), radius of gyration (Rg),
often restricted to the determination of simple geometric parame- hydrated particle volume (Vp) and maximum particle diameter
ters. A renaissance in the study of such systems by structural biol- (Dmax). The Guinier analysis developed by A. Guinier in the 1930s
ogists over the last 5–10 years has led to the development of novel (Guinier, 1939) is still the most straightforward method for the
approaches for the analysis of flexible systems including multi-do- extraction of the forward scattering intensity I(0) and the radius
main and intrinsically unfolded proteins (Bernado et al., 2005, of gyration, Rg. For a monodisperse solution of globular macromol-
2007; Obolensky et al., 2007). ecules the Guinier equation is defined as:
130 H.D.T. Mertens, D.I. Svergun / Journal of Structural Biology 172 (2010) 128–141

1 tive or repulsive inter-particle effects and sample polydispersity
IðsÞ ¼ Ið0Þ exp R2g s2 ð2Þ
3 result in deviations from linearity (Fig. 2A and B). For example,
samples that contain a significant proportion of non-specific aggre-
In principle, I(0) and Rg can be extracted from the y-axis inter-
gates yield scattering curves and Guinier plots with a sharp in-
cept and the slope of the linear region of a Guinier plot (ln[I(s)] ver-
crease in intensity at very small values of s, while samples
sus s2), respectively (Fig. 2A and B). However, the range (smin to s1)
containing significant inter-particle repulsion yield curves and
over which the Guinier approximation is valid for each measured
Guinier plots that show a decrease in intensity at small values of
scattering curve must be considered. The lower limit of this range,
s. Note that it might still be possible to obtain ‘‘linear fits” even
smin, is usually restricted by the experimental set-up, and for an
to data with significant concentration/aggregation effects
ideal sample is taken to be the minimum angle for which intensity
(Fig. 2B, fits 1 and 3), albeit in rather short ranges, and care must
is recorded. The Guinier approximation is based on a power law
be taken to avoid wrong results caused by these effects. Moreover,
expansion, used to describe the linear dependence of ln[I(s)] on
even a ‘‘long” linear Guinier plot does not always guarantee that a
s2 (Guinier, 1939). When extended to larger values of s, the higher
sample is monodisperse and researchers are advised to check sam-
order terms in the expansion begin to significantly contribute to
ple monodispersity using methods such as dynamic light scatter-
the scattering intensity, breaking this linear dependence. Given
ing before conducting SAXS measurements.
that the power value (sRg)n decreases with n for sRg < 1 and in-
Samples often contain molecules that interact strongly with
creases for sRg > 1, s1 < 1/Rg is a reasonable estimate for the upper
each other in a concentration dependent manner. In non-ideal
limit of the Guinier fit. However, it is often the case that the range
solutions, strong attractive or repulsive inter-particle interactions
smin < s < 1/Rg contains too few points, especially in the case of very
modulate the recorded scattering intensity particularly at low an-
large macromolecules. It is common in biological SAXS to extend
gles (s < 1 nm1) and influence the parameters extracted from the
this range up to s1 < 1.3/Rg, so that a sufficient number of data
SAXS curve. For example, attractive interactions can result in the
points are available for the estimation of I(0) and Rg. Practice shows
overestimation of I(0) (and thus MM) and Rg and repulsive interac-
that 1.3/Rg is a safe estimate for s1, which does not introduce sys-
tions can result in these parameters being underestimated. The
tematic deviations from linearity.
contribution of these interactions to the scattering intensity can
A non-linear Guinier plot is a strong indicator of poor sample
be separated from that derived from the shape of the particles by
quality. Improper background subtraction, the presence of attrac-
Fig. 2. Standard plots for characterization by SAXS. (A and B) SAXS curves and Guinier plots for BSA samples measured at X33 (DORIS, Hamburg) in different buffers showing
(1) aggregation, (2) good data and (3) inter-particle repulsion. The Guinier fits for estimation of Rg and I(0) are displayed, with the linear regions defining smin and smax used for
parameter estimation indicated by the thick lines. (C and D) SAXS curves and Kratky plots for lysozyme samples measured at X33 (DORIS, Hamburg) showing (1) folded
lysozyme, (2) partially unfolded lysozyme (in 8 M urea), (3) partially unfolded lysozyme at 90 °C and (4) unfolded lysozyme (in 8 M urea at 90 °C). Plots are arbitrarily
displaced on the vertical axis for clarity with the exception of (D), where all curves have been scaled to the same forward scattering intensity, I(0).
Z 1
recording scattering patterns at several concentrations. From a r2 sin sr
pðrÞ ¼ s2 IðsÞ ds ð4Þ
concentration series it is usually possible to identify a sample con- 2p2 0 sr
centration where inter-particle interactions are negligible or to
The distance distribution function is a real space representation
extrapolate the data to infinite dilution to obtain an ‘‘ideal” curve
of the scattering data and allows one to graphically display the
for further structural analysis. In addition, characterization of the
peculiarities of the particle shape (Fig. 3). For example, globular
concentration dependent behavior of the sample provides an
particles yield bell-shaped profiles with a maximum at approxi-
important source of information that has been used to determine
mately Dmax/2 and multi-domain particles often yield profiles with
interaction potentials between macromolecules in solution
multiple shoulders and oscillations corresponding to intra and
(Tardieu et al., 1999). This can help to define conditions for crystal-
inter-subunit distances. Computation of p(r) is not straightforward
lization, which typically require weakly attractive interactions (for
as a limited range of I(s) is available (from smin to smax), and direct
detailed reviews on the determination of interaction potentials
Fourier transformation of the scattering curve from this finite
from SAXS data see (Koch et al., 2003) and (Finet et al., 2004).
number of points is not possible. A solution to this problem is
With knowledge of the limitations of the Guinier approximation
the indirect Fourier transformation method first proposed by O.
in mind, the method is an essential first step in sample character-
Glatter in the 1970s (Glatter, 1977). In this approach p(r) is repre-
ization by SAXS. From visual inspection of the Guinier plot samples
sented as a linear combination of K orthogonal functions uk, in the
can be screened for non-specific aggregation (Fig. 2A and B, curve
range [0, Dmax], with Dmax being a user defined variable:
1), the presence of inter-particle repulsion (Fig. 2A and B, curve
3), the prevalent oligomerisation state in solution estimated and X
K
the invariant shape and weight parameters, Rg and I(0) extracted. pðrÞ ¼ ck /k ðsi Þ ð5Þ
In the past, Guinier analysis was always done interactively; re- k¼1
cently, automated procedures have become available. In particular,

The optimal coefficients ck are sought through minimization of the
the program AUTORG (Petoukhov et al., 2007) employs statistical
functional:
methods to optimize the range of s, to detect possible aggregation
or repulsive interactions and, based on this, to evaluate the quality U ¼ v2 þ aPðpÞ ð6Þ
and reliability of the extracted parameters.
Following on from the Guinier analysis the MM can be esti- where the first term, v2 is the goodness of fit between the experi-
mated based on knowledge of the forward scattering intensities mental data and that calculated by the direct transform of the p(r)
and concentrations of both the macromolecule of interest and a function (Eq. (7)), and the second (penalty) term, P(p) ensures the
standard such as bovine serum albumin. This estimate requires smoothness of the p(r) function (Eq. (8)).
normalization against the solute concentrations for the two mea- N 2
1 X Iexp ðSj Þ cIcalc ðSj Þ
surements, and the accuracy of the MM estimate is limited v2 ¼ ð7Þ
(Mylonas and Svergun, 2007). An alternative approach to MM N 1 j¼1 rðSj Þ
estimation that is more suited to solutions with significant lipid,
carbohydrate or nucleic acid content (including for example, glyco- Z Dmax
2
proteins or protein–lipid complexes) involves the use of water as a PðpÞ ¼ ½p0 dr ð8Þ
0
standard (Orthaber et al., 2000).
Independent from the Guinier analysis, the hydrated particle where N, r and c are the number of data points, the standard devi-
volume (Vp) can be obtained from the data on a relative scale, ations and scaling factor respectively. The regularizing multiplier a
avoiding inaccuracies in parameter estimation caused by errors balances between the fit to the data and the smoothness of the p(r).
in concentration measurement. Assuming a uniform electron den- One should note here that fitting the experimental data using Eq.
sity inside the particle, Vp is estimated following Porod’s equation (6) is often employed in physical experiments including SAXS data
(Porod, 1982): analysis and interpretation. As will be presented below, ab initio and
Z 1 rigid body modeling methods will use a similar notion of a penalty
V p ¼ 2p2 Ið0Þ=Q ; Q¼ s2 IðsÞ ds ð3Þ term to ensure that physically sensible solutions are obtained.
0
In the indirect transform program GNOM (Svergun, 1992), the
where Q is the so-called Porod invariant. solution yielding the p(r) function is evaluated using perceptual
For real macromolecules the electron density is of course not criteria, providing the user with the means to easily identify reli-
uniform, however, at sufficiently high MM (>30 kDa), the subtrac- able solutions and to obtain an optimal value of Dmax. This proce-
tion of an appropriate constant from the scattering data generates dure has also been automated in the program AUTOGNOM
a reasonable approximation to the scattering of the corresponding (Petoukhov et al., 2007), where multiple GNOM runs are performed
homogenous body. The particle volume, Vp allows one to make an without user intervention across a range of Dmax values. The
alternative estimate of the MM with the added advantage that parameters estimated from the indirect Fourier transform ap-
this estimate is independent of errors in the sample concentra- proach, I(0) and Rg are typically more accurate than those obtained
tion. Typically, for a globular protein Vp (in nm3) is 1.5–2 times from a Guinier analysis as the entire scattering curve is used for
the MM (in kDa). While this estimate is a crude approximation their estimation.
only, with prior knowledge of the expected size/state of the sam- For the study of protein folding the Kratky plot (s2I(s) vs s) can
ple, a rough indication of the homogeneity of the sample can in be used as an indication of the folded/unfolded state (Doniach,
this way be made available directly following measurement. A 2001). Folded globular proteins typically yield a prominent peak
Web server has recently been made available allowing one to at low angles (Fig. 2C and D, curve 1), whereas unfolded proteins
estimate the MM from SAXS data based on this approach (Fischer show a continuous increase in s2I(s) with s (Fig. 2C and D, curve
et al., 2010). 4). Flexible multi-domain proteins can also potentially be identi-
Due to the limitations of the Guinier approximation, the extrac- fied from the Kratky plot, displaying a mixture of characteristic fea-
tion of Rg and I(0) from scattering data is also routinely done tures of both folded and unfolded proteins similar to that observed
through the use of indirect Fourier transform methods. Fourier for a partially unfolded state (Fig. 2C and D, curves 2 and 3). How-
transformation of the scattering intensity yields the distance distri- ever, recent studies (Bernadó, 2009) show that flexible proteins can
bution function, p(r): yield Kratky plots that do not indicate the presence of flexibility
Fig. 3. Scattering intensities and distance distribution functions, p(r) calculated for typical geometric shapes: Solid sphere (black), prolate ellipsoid (red), oblate ellipsoid
(blue), two-domain (green) and long rod (cyan). The bead models used for the calculation of scattering intensities are shown above the plots.
but suggest relatively rigid compact globular structures (see Sec- lows for rapid analytical computation of scattering patterns
tion 6). from known structures. This expression is readily incorporated
Rapid sample characterization under near native solution condi- into algorithms for the minimization of discrepancy v between
tions is one of the major advantages of SAXS over other structural experimental and calculated scattering curves (Eq. (7)). The
techniques. Overall parameters describing size, shape and volume spherical harmonics formalism is heavily exploited by most ad-
can be extracted from SAXS patterns almost immediately following vanced modeling programs.
measurement and used to answer important biological questions. In the initial ab initio approach, the shape of particles was de-
However, in addition to sample characterization from extracted scribed by an angular envelope function, the latter developed into
parameters only, characterization of the three-dimensional struc- a series of spherical harmonics (Stuhrmann, 1970a). This method
ture of the macromolecule or complex from the scattering curves was further developed into the first publicly available program
is also possible. One approach is to use the SAXS profile to search a SASHA (Svergun et al., 1996). While this method was a major
database of calculated scattering curves based on deposited struc- breakthrough in the determination of low-resolution structure
tures in the PDB, with the aim of finding models that describe the from SAXS it was restricted to particles without internal cavities.
measured data. In a recent high throughput study the DARA database More detailed ab initio reconstructions became possible through
(Sokolova et al., 2003) was successfully used to find closely matching the development of automated bead-modeling. This approach was
structures of a series of proteins measured by SAXS (Hura et al., first proposed by Chacon et al. (1998)) and further implemented in
2009). A more direct approach is to take advantage of modern meth- different variations by other authors (Bada et al., 2000; Chacon
ods of ab initio reconstruction from scattering data and combine this et al., 2000; Svergun, 1999; Vigil et al., 2001; Walther et al.,
information with other complementary structural and biochemical 2000). The most popular ab initio bead-modeling program in
data. The following two sections focus on the recent developments current use is DAMMIN (Dummy Atom Model Minimisation)
in the determination of structure from SAXS. (Svergun, 1999). The algorithm represents a particle as a collection
of M (>>1) densely packed beads inside a constrained (usually
3. Ab initio methods spherical) search volume, with a maximum diameter defined by
the experimentally determined Dmax (Fig. 4). (2) Each bead is ran-
The reconstruction of low-resolution 3D models from SAXS data domly assigned to the solvent (index = 0) or solute (index = 1), and
alone is now a standard procedure and as such can also be consid- the particle structure is described by a binary string X of length M.
ered a rapid characterization tool. The basic principles behind The shape reconstruction is conducted starting from a random ini-
shape determination from 1D SAXS data were established in the tial approximation by simulated annealing (SA) (Kirkpatrick et al.,
1960s, where scattering patterns were computed from different 1983), minimizing the goal function as defined in Eq. (6). The dis-
geometrical shapes and compared with experimental data. These crepancy (v2) is evaluated in Eq. (7) between the experimental and
trial-and-error methods were superseded in the 1970s through calculated scattering intensities (the latter being rapidly computed
the introduction of a spherical harmonics representation by Stuhr- using spherical harmonics). At each step in the SA procedure the
mann (1970b). In this representation a multipole expansion is used assignment of a single bead is randomly changed leading to a
in order to derive a simple expression for the SAXS intensity I(s): new model X0 . The solution is constrained by the penalty term,
P(X), requiring that the beads must be connected and the model
X
1 X
l
2
compact to ensure that physically feasible low-resolution struc-
IðsÞ ¼ 2p2 jAlm ðsÞj ð9Þ tures are generated. A multiphase version of DAMMIN bead-mod-
l¼0 m¼l
eling is implemented in the program MONSA (Svergun and
This expression, which is a sum of independent contributions Nierhaus, 2000), also widely used when contrast variation data
from the substructures corresponding to different spherical har- from SANS and/or multiple SAXS curves from components of a
monics (where Alm(s) are the partial scattering amplitudes), al- complex (e.g. a nucleoprotein complex) are available.
scattering curves can only be fit within a restricted range (typically

up to s 2.5 nm1, i.e. a resolution of 2.5 nm, where resolution d is
defined as d = 2p/s). An alternative approach for proteins is to rep-
resent the molecule not as a collection of uniformly distributed
beads, but as an assembly of dummy residues (DR). In this simpli-
fication of the atomic structure a globbic approximation is em-
ployed, where the atomic scattering densities for the atomic
groups of each amino acid are combined to generate an effective
scattering density of an average amino acid in water (Guo et al.,
1995, 1999; Harker, 1953). Such an approach is used in the pro-
gram GASBOR (Svergun et al., 2001), where the DR scattering rep-
resents the scattering from an amino acid averaged over the
abundance of amino acids in proteins. The program starts from a
randomly distributed ‘‘gas” of DRs in a spherical search volume de-
fined by Dmax. The number of DRs is equal to the number of resi-
dues in the protein sequence, and their positions are determined
by SA driven minimization using Eq. (6), with a penalty term
requiring that the DRs form a protein-like or folded chain-compat-
ible structure. The latter conditions are ensured by requiring, in
particular, that the average distance histogram of neighboring
DRs in the model is similar to that for globular proteins. As the lim-
itation of particle homogeneity is abrogated the data can be fit to
much higher angles than for the bead-modeling programs (up to
s < 10 nm1). GASBOR is routinely used by structural biologists to
determine the low-resolution structures of proteins and protein
complexes (Chen et al., 2007; De Marco et al., 2009; Pavkov
et al., 2008; Schmidt et al., 2010; Trindade et al., 2009). In the field
of structure based development of protein therapeutics GASBOR
was recently used to generate low-resolution ab intio models of
PEGylated Haemoglobin (Svergun et al., 2008). In combination
with multiphase modeling using MONSA the structures deter-
mined help explain how the increased vascular retention of such
therapeutics is likely a result of the surface shielding and intermo-
Fig. 4. Ab initio modeling procedure using DAMMIN. (A) Starting from a spherical lecular repulsion associated with PEG conjugation.
search volume a fitting procedure is conducted until a final model is generated One must note that ab initio methods, seeking to reconstruct a
satisfying not only a fit to the experimental data, but also forming a compact and 3D shape from a 1D scattering pattern cannot provide a unique
connected model of dummy atoms/beads. (B) The spherical search volume with
solution and, when running the programs multiple times, some-
beads assigned to the particle (yellow) and solvent (blue).
what different models are obtained. A comparison of these models
ensures that the most persistent features are identified from a
Although DAMMIN is reasonably fast, typically taking several number of models that fit the data equally well but show variation
minutes to several hours on modern computers, the widespread in shape. Thus the intrinsic ambiguity of SAXS data interpretation
use of the program for high-throughput data analysis necessitates can be reduced and reliable average models obtained. The average
that, where possible, improvements in speed and performance ab initio SAXS model is conceptually similar to a mean average
should be sought. Thus DAMMIN has recently been re-written structure from an NMR ensemble, albeit at a much lower resolu-
and optimized for performance. The new implementation is the tion. The programs SUPCOMB (Kozin and Svergun, 2001) and
program DAMMIF (Franke and Svergun, 2009) where F refers to DAMAVER (Volkov and Svergun, 2003) have been developed for
fast. this purpose. SUPCOMB performs the rapid superposition of
DAMMIF differs in several important respects to DAMMIN. (1) ensembles of models and calculates the degree of structural simi-
The bounded search volume has been replaced by an unrestricted larity. Further, SUPCOMB identifies the most probable/representa-
volume that can grow in size as needed during the SA procedure. tive ensemble member and DAMAVER averages the superposed
This improvement helps to avoid artefactual boundary effects that models over the ensemble yielding a smoothed model containing
may occur when using a search volume restricted by a slightly the most persistent features in the reconstructions. The use of
underestimated Dmax. (2) Prior to the calculation of scattering these methods allows for the assessment of the uniqueness of
amplitudes models that are disconnected are immediately re- the ab initio models determined from scattering data, but a re-
jected, and the calculation is performed only for interconnected searcher must still critically evaluate the chosen model based on
models. (3) The necessary scattering amplitudes in terms of spher- the expected size and hydrated volume (with possible multimers
ical harmonics are intelligently pre-computed for each bead con- also in mind). Finally, one must always remember that the scatter-
tributing to the total scattering at least once. All these measures ing patterns are invariant to handedness such that an enantiomor-
accelerate the modeling procedure by 25–40 times compared to phic model must always be considered (SUPCOMB/DAMAVER do
the original DAMMIN, and already a number of publications are allow for this enantiomorphism).
appearing in the literature citing the use of DAMMIF (Cheng The ab initio analysis of proteins and protein complexes with
et al., 2009; Heikkinen et al., 2009; Schmidt et al., 2010; Yadavalli SAXS is very usefully complemented by information from other
et al., 2009). methods. High-resolution crystal and NMR structures can be
The resolution of shape determination from bead models and docked into the low-resolution SAXS models, and the shapes
envelope functions is limited by the assumption of a uniform elec- provided by electron microscopy (EM) can be used as starting vol-
tron density distribution within the particle, and consequently umes for bead-modeling (Svergun, 1999). Information regarding
expected symmetry and anisometry can be particularly useful for mer. From the fit of the theoretical scattering curves computed
obtaining reliable ab initio models, and can also significantly speed with the program CRYSOL (Svergun et al., 1995) to the SAXS data
up the computations. Symmetry restrictions associated with the (Fig. 5), the heterohexamer was identified as the correct model in
space groups P2–P12, P222–P62, cubic and icosahedral symmetry solution.
can be explicitly defined in DAMMIN and GASBOR, and most of New approaches are currently being developed, particularly for
these symmetries are also applicable in DAMMIF and MONSA. the accurate prediction of wide-angle scattering data (WAXS)
The use of correct symmetry allows one to further restrain the where information on both tertiary and secondary structure can
solution to yield more detailed ab initio models; however, symme- potentially be extracted (Bardhan et al., 2009; Park et al., 2009).
try must always be employed with caution and models in P1 These approaches also include alternative methods for the approx-
should also be calculated for control and comparison. imation of the hydration layer, including the addition of an explicit
layer of water molecules in a solvent density matching that of the
4. Computation of scattering from high-resolution models bulk solution (Yang et al., 2009).
Another method for the rapid characterization of proteins and 5. Rigid body modeling
complexes if high-resolution structures or homology models are
known is the computation of scattering curves from atomic mod- The assembly of macromolecular complexes can be studied
els, and the comparison of these predicted curves with the exper- through the docking of individual components into ab initio shapes
imentally determined SAXS profiles (Hough et al., 2004; King et al., (Wriggers and Chacón, 2001). However, as the resolution of SAXS
2005; Vestergaard et al., 2005). Given a model, the theoretical scat- derived shapes is low, it is more reliable to model the assembly
tering curve can be computed and fit to the measured data, with of such complexes through direct refinement against the scattering
this computation taking into account the atomic scattering in va- data. A number of interactive and automated approaches have
cuo, the excluded volume (the volume occupied by the biomolecule been developed using SAXS to determine the positions and orien-
in solution that is inaccessible to solvent) and scattering from the tations of subunits within macromolecular complexes (Boehm
hydration layer. A model that provides a good fit to the data is con- et al., 1999; Konarev et al., 2001; Petoukhov and Svergun, 2005;
sidered a valid description of the structure under the solution con- Sun et al., 2004). From the known structures (or homology models)
ditions used for the measurement. of subunits, the theoretical scattering of a complex can be rapidly
A number of methods exist for the calculation of theoretical scat- calculated using an application of the spherical harmonics formal-
tering curves from atomic models, these methods generally differ in ism mentioned in the Section 3. This formalism, the basis for the
their approach to the calculation of the atomic scattering intensity, above described programs CRYSOL (Svergun et al., 1995) and CRY-
the subtraction of solvent excluded volume and the way in which SON (Svergun et al., 1998) is applied to automated rigid body mod-
a hydrated surface with a solvent density higher than that of the bulk eling in the programs SASREF and BUNCH (Konarev et al., 2006;
solvent is approximated. Traditionally, the Debye formula (Debye, Petoukhov and Svergun, 2005).
1915) is used for the calculation of atomic scattering, however, the SASREF is a comprehensive automated rigid body modeling pro-
time required for computation scales quadratically with the number gram for SAS, allowing for the simultaneous fitting of multiple
of atoms and makes the method less useful for large proteins and scattering curves (e.g. when multiple constructs such as deletion
complexes. Recent programs employ the globbic approximation mutants have been measured and also for contrast variation data
(see Section 3) to speed up computation using the Debye formula from SANS). The use of symmetry, orientational constraints (e.g.
(Yang et al., 2009). Arguably, the most efficient approach to the cal- from residual dipolar couplings measured by NMR), inter-residue
culation of the scattering intensity from atomic models is the spher-
ical harmonics approximation used in the programs CRYSOL
(Svergun et al., 1995) for SAXS and CRYSON (Svergun et al., 1998)
for SANS. In this approach the computation time scales linearly with
the size of the model and it is highly accurate up to s < 5.0 nm1, but
has been successfully used up to much higher resolutions (up to
s = 10–15 nm1). CRYSOL/CRYSON employ a gaussian sphere
approximation for the calculation of the excluded volume (Fraser
et al., 1978) and use spherical harmonics to calculate an envelope
at the surface of the atomic model to approximate the hydration
layer. For prediction of the scattering intensity of theoretical models
default parameters for the excluded volume and the excess scatter-
ing density of the hydration layer are used. It is assumed that the
scattering density of the hydration layer is 10% greater than that
of the bulk, and this assumption has been verified experimentally
in a combined SAXS/SANS study (Svergun et al., 1998). When used
in fitting mode, the excess scattering density of the hydration layer
is used as a fitting parameter and adjusted to best fit the scattering
data up to a resolution of about 0.5 nm.
Computation of scattering from high-resolution models is often
used to identify the biologically active conformations of crystal
structures and help distinguish between alternative crystallo-
graphic dimers and/or higher oligomers (De Marco et al., 2009;
Santiago et al., 2009). For example, a new crystallographic form Fig. 5. Identification of heterohexameric solution state of the human Cdt1–Geminin
of the protein complex of Cdt1 and Geminin was validated in solu- complex by SAXS (De Marco et al., 2009). Experimental scattering pattern for the
Cdt1–Geminin complex in solution and the fits calculated from the crystal
tion by SAXS (De Marco et al., 2009). In this study a heterohexa- structures of the heterotrimer (red broken line) and heterohexamer (blue broken
meric structure was observed in the crystal whereas a previous line). It is clear that the heterohexamer (blue structure) fits the experimental data
crystallographic study had shown only the existence of a heterotri- while the heterotrimer (red structure) does not.
contacts (e.g. from mutagenesis or cross-linking experiments) and applied to the larger two constructs. The NMR ensemble of con-
inter-subunit distances (e.g. from FTIR and FRET) are fully sup- formers determined for fH12–13 was independently validated
ported. Starting from an arbitrary positioning of subunits, subject through a good fit to the SAXS data and the near-perfect superpo-
to any user defined constraints SASREF conducts a series of random sition of the ab initio structure with the ensemble (Fig. 6A and B).
rigid body movements and rotations, using SA to search for a best The rigid body models of fH11–14 and fH10–15 were highly repro-
fit of the computed complex scattering to the experimental data. ducible, forming zig-zag arrangements, and demonstrated that the
The target to be minimized has the form of Eq. (6) combining the core of fH is compact (Fig. 6A and B).
discrepancy (possibly calculated over multiple scattering patterns)
and penalty term. The latter may include restraints from other
methods such as those mentioned above, but always incorporates 6. Flexible systems
constraints making sure that the models generated are intercon-
nected and have no main-chain/backbone steric clashes (side- In the last example of the preceding section the scattering data
chains of proteins are ignored). SASREF is actively used by many from a multi-domain protein were successfully analyzed in terms
groups; a recent example is the determination of the quaternary of rigid body models, assuming therefore that all linkers were rigid
structure of the Drosophila neuronal adhesion protein Amalgam such that constructs displayed no flexibility in solution. It was in-
(Zeev-Ben-Mordehai et al., 2009). From this study a V-shaped di- deed the case for this particular protein (and it was possible to
mer with a parallel arrangement of monomers was identified, demonstrate this, see below), but very often in practice one deals
and along with the identification of the dimerization interface pro- with systems, which possess significant flexibility in solution.
vided a structural mechanism for the observed adhesion of this SAXS was proven to be a powerful technique for the analysis of
protein to neuronal cells. In another example, the low-resolution flexible systems (Bernadó et al., 2007, 2005; von Ossowski et al.,
structure of the N-terminal region of the enzyme poly(ADP-ribose) 2005). However, a recent study by Bernadó (2009) clearly presents
polymerase-1 (PARP-1) was determined by SAXS (Lilyestrom et al., the difficulties associated with meaningful interpretation of SAXS
2010). In this study, both ab initio modeling using DAMMIN and ri- curves for highly flexible modular proteins. This study demon-
gid body modeling with SASREF were used to identify the extended strates that proteins may be wrongly identified as rigid from
modular s-shaped structure of the N-terminal region of PARP-1. dynamically averaged SAXS profiles and that several indicators
This model, combined with DNA binding studies were used to pro- for inter-domain flexibility should be monitored. Typically, ab ini-
pose that the mechanism for activation of PARP-1 involves a con- tio models produced from dynamically averaged scattering data
formational rearrangement upon the binding of damaged DNA display a decrease in resolution or structural detail, while rigid
and not dimerization. body models are generated with highly extended conformations
SASREF requires that complete high-resolution models (or reli- and a paucity of inter-domain contacts. A recently developed
able homology models) of all of the subunits are available for the ensemble optimisation method (EOM) (Bernadó et al., 2007) pro-
rigid body modeling of a protein complex. When the structures vides a useful approach for assessing the flexibility of the system
of linkers or entire domains are unknown an alternative approach under study when high-resolution structures of domains are
combining both rigid body modeling and ab initio methods is used. available.
The program BUNCH (Konarev et al., 2006; Petoukhov and Svergun, Both intrinsically unfolded proteins and modular multi-domain
2005) uses DRs to model missing regions in both protein com- proteins with flexible linkers can be represented as ensembles of
plexes and multi-domain proteins connected by flexible linkers. structures. In EOM a large pool of random configurations is gener-
As in SASREF a SA minimization is used to locate a best fitting ated and ensembles are selected from this pool using a genetic
arrangement of the rigid bodies and also the optimal local confor- algorithm, such that the average computed scattering over the
mation of DRs. This approach has been used to successfully deter- ensemble fits the experimental scattering data (Bernadó et al.,
mine the structures of multi-domain proteins with flexible linkers 2007). If the Rg distribution of the models in the selected ensem-
(Clantin et al., 2010; Gut et al., 2009; Kozlov et al., 2009; Nemeth- bles is as broad as that in the initial random pool, the protein is
Pongracz et al., 2007; Schmidt et al., 2010), and also for the addi- likely to be flexible; obtaining a narrow Rg peak suggests that the
tion of missing portions to crystal structures (Gut et al., 2009). system may be rigid. The EOM results provide a useful guidance
Rigid body modeling using SAXS data is very actively employed but to reach definitive conclusions regarding the flexibility of the
by structural biologists for the analysis of macromolecular com- system these data should be correlated with complementary tech-
plexes. For example, the combined ab initio and rigid body model- niques including e.g. NMR relaxation studies. In fact, the above
ing approach in BUNCH was successfully used to determine the data from the human complement factor H was analyzed using
compact architecture of the central portion of the human comple- EOM to suggest the relatively rigid nature of the constructs studied
ment factor H (fH) (Schmidt et al., 2010). fH is a modular multi-do- (Fig. 6C), supporting the hypothesis that the central domains of this
main protein composed of 20 complement control protein modules protein do not provide a flexible tether between N and C-terminal
(CCPs; each 60 residues) connected by short linkers and is a ma- ligand binding sites in agreement with the NMR data.
jor component of complement regulation in the human innate im- A recent application of the ensemble representation of flexible
mune system. High-resolution structures of 12 terminal fH CCPs structures is the combined NMR and SAXS study of inter-domain
have been previously determined in isolation or as two to four do- flexibility in full-length matrix metalloproteinase-1 (MMP-1)
main fragments (reviewed in (Schmidt et al., 2008)), but the struc- (Bertini et al., 2009). In this study it was shown that a dynamic
ture of the full length protein is unknown. Previous studies by equilibrium of open and closed conformations of MMP-1 are pres-
transmission electron microscopy, analytical ultra centrifugation ent in solution, with the EOM analysis supporting the NMR data.
(AUC) and SAXS indicate that full length fH is not fully extended The Rg distribution of the generated random pool of conformations
in solution and may contain highly flexible linkers, allowing the is broad, covering a range of compact and extended structures
molecule to fold back upon itself (Aslam and Perkins, 2001). SAXS (20 to 45 Å). However, the Rg distribution of the selected ensem-
data was collected on several deletion mutants consisting of the ble of structures displays a relatively sharp peak at 25 Å (Fig. 7),
central CCPs 10–15, and the NMR structure of CCPs 12–13 suggesting that the most highly populated conformations of MMP-
(fH12–13) determined in the same study (Schmidt et al., 2010). 1 are compact. An extended tail is observed in the distribution at
The constructs fH12–13, fH11–14 and fH10–15 were determined higher values of Rg, suggesting that the MMP-1 is inherently flexi-
to be monomeric in solution and rigid body modeling with BUNCH ble in solution and that a population of extended conformations
Fig. 6. SAXS study of human factor-H (Schmidt et al., 2010). (A) Scattering curves for three constructs of the central 2–5 CCP modules of factor-H. Broken lines represent fits
obtained by CRYSOL for the best fH12–13 NMR model, or by rigid body modeling (BUNCH) for fH11–14 and fH10–15; curves have been arbitrarily displaced along the
logarithmic axis for clarity. (B) Overlay of the NMR ensemble of fH12–13, and the rigid body models (BUNCH) for fH11–14 and fH10–15, with ab initio shape envelopes
produced with DAMMIF. (C) Radius of gyration distributions of pools (broken lines) and selected structures (coloured areas) for the EOM analysis of fH12–13, fH11–14 and
fH10–15. Each distribution is skewed toward either extended (fH12–13) or compact (fH10–14 and fH10–15) structures.
assembly process), a model-independent estimate of K can be

obtained using singular value decomposition (SVD) (Golub and
Reinsh, 1970; Konarev et al., 2006). In the program OLIGOMER
(Konarev et al., 2003), volume fractions are readily computed pro-
vided the scattering patterns of the components are known (e.g.
provided by CRYSOL from known structures of components). OLI-
GOMER has been successfully used to characterize oligomeric
equilibria and complex formation (Bernadó et al., 2009; Niemann
et al., 2008; Paravisi et al., 2009), and has been applied to the study
of self fibrillating proteins (Vestergaard et al., 2007). Recently, a
multivariate curve resolution (MCR-ALS) method (Blobel et al.,
2009) was proposed to determine scattering patterns from compo-
nents in oligomeric mixtures.
An exciting development in the analysis of mixtures from scat-
tering data is the determination of low-resolution ab initio models
of protein–protein complexes that exist as minority species in
solution. In a recent study reliable models of a major and minor
component from a monomer–dimer equilibrium could be recon-
structed from the deconvolution of SAXS data (Blobel et al.,
2009). In this work, Blobel et al. analyzed a solution of low molec-
ular weight phosphatase (lmwPTP) using a multivariate curve res-
olution method (MCR-ALS) to characterize the monomer–dimer
equilibrium. From the extracted SAXS contributions corresponding
to the monomeric and dimeric components (the dimer component
Fig. 7. EOM analysis of MMP-1 (Bertini et al., 2009). Radius of gyration distribution constituting only 15% of the total protein concentration), DAM-
of the pool (broken line) and selected (coloured area) structures for the EOM
MIN models were generated and were in very good agreement
analysis of MMP-1. The crystal structure of MMP-1 is shown inset.
with the corresponding monomer and dimer crystal structures
(Fig. 8).
also exist. In another example, tetrameric Flavorubredoxin (FDP), Another powerful method for the analysis of mixtures is time-
the rigid body models generated using SASREF and defined P222 resolved SAXS (TR-SAXS). TR-SAXS has been used for the study of
symmetry suggested that a highly flexible linker between the core protein and RNA folding (Cammarata et al., 2008; Lamb et al.,
structure and c-terminal rubredoxin (Rd) domains was likely 2008a), the formation and dissociation of complexes and for
(Petoukhov et al., 2008). To investigate the degree of flexibility kinetic analyses of conformational change stimulated by some
and to quantitatively describe the preferential arrangement of external stimulus (Cammarata et al., 2008; Lamb et al., 2008b).
the Rd domains an EOM analysis was conducted. Indeed, through While manual mixing and a series of static measurements can be
the addition to the random pool of conformations generated with conducted for slow (minutes to hours) molecular processes, stud-
constraints maintaining extended linkers, an excellent fit to the ies of sub-millisecond to millisecond processes (for example pro-
data was obtained. In all selected models no contacts between tein folding under near native conditions of pH and temperature)
the FDP core and Rd domains were observed and all linkers were require devices for rapid mixing and a high-brilliance X-ray beam
significantly extended and peripherally located. coupled with a state-of-the-art detector. Third-generation syn-
As a word of caution it should be mentioned that EOM analysis chrotrons provide the necessary flux for recording SAXS data with
of polydisperse samples containing mixtures of oligomers or aggre- good signal to noise from very short (millisecond) exposures, and
gates may provide an erroneous indication of sample flexibility and modern fast read-out detectors are now in use or a being installed
lead to false conclusions. Similar to the ab initio and rigid body
modeling methods, careful sample characterization prior to using
this method is essential (see Section 2).
7. Analysis of mixtures
Another important application of SAXS to rapidly characterize

protein solutions is the quantitative description of mixtures (e.g.
oligomeric equilibria and assembly processes). For mixtures and
polydisperse solutions of non-interacting particles the resulting
scattering pattern is a sum of the contributions from each compo-
nent of the mixture Ik(s), weighted by the volume fraction vk of that
component:
X
K
IðsÞ ¼ v k Ik ðsÞ; ð10Þ
k¼1
Several methods have been developed to help simplify the anal-

ysis of equilibrium mixtures using SAXS (Feigin and Svergun, 1987;
Fowler et al., 1983; Koenig et al., 1992; Konarev et al., 2003). If the Fig. 8. Superposition of the crystal structures of lmwPTP monomer (A) and dimer
(B) with the ab initio DAMMIN models generated from analysis of the oligomeric
number of components K in the mixture is not known but a series equilibrium (Blobel et al., 2009). Note that the ab initio model of the dimer is
of measurements is available from the samples containing differ- affected by boundary effects caused by the limited search volume. Such artifacts are
ent amounts of the components (e.g. at different stages of an absent in DAMMIF, where an adaptable search volume can be used (see Section 3).
at many SAXS beamlines, making time-resolved scattering studies ble to the determination of DNA and RNA structures by NMR
more available to researchers. The recent time-resolved wide-an- (Grishaev et al., 2008b; Schwieters and Clore, 2007).
gle scattering (WAXS) study of the conformational changes of hae- SAXS data can be used to complement many studies in struc-
moglobin highlights the power of the technique (Cammarata et al., tural biology including validation of high-resolution models, solv-
2008). In this study, nanosecond time resolution was made possi- ing the phase problem in crystallography and the refinement of
ble through the laser-induced photolysis of the carbon monoxide solution structures. New approaches to structural modeling are
ligand, allowing the transition of the protein from the R to the T currently being developed which seek to incorporate as much com-
state to be followed in solution. TR-SAXS/WAXS has a bright future plementary data as possible. The integrated modeling platform
in modern structural biology as the study of kinetic processes can (IMP) is one such project using SAXS data as an additional spatial
help to link structure with biological function, especially for com- restraint (Forster et al., 2008).
plex systems of interacting molecules.
9. Future developments
8. Combining NMR, crystallography and SAXS
The study of biological systems using solution SAXS is increas-
The combination of X-ray crystallography and SAXS as comple- ingly gaining momentum, with many research groups looking to
mentary methods is very well established. The use of SAXS to incorporate this technique into their research programs. As most
investigate the solution properties of crystal structures was pio- of the SAXS analysis tools have now reached a mature state, their
neered in the 1970s and early 1980s, with the development of application is straightforward and can even be performed auto-
sophisticated methods for the prediction of theoretical scattering matically. Therefore, not only evaluation of the overall parameters,
from crystal structures and initial attempts at rigid body modeling but also shape determination, analysis of the oligomeric composi-
(Fedorov and Denisyuk, 1978; McDonald et al., 1979; Pavlov, tion and to some extent rigid body modeling of quaternary struc-
1985). These methods have been actively developed and now pro- ture can be considered rapid characterization tools for proteins
vide powerful tools used by structural biologists to compliment and complexes (Fig. 9). Having said that, one should keep in mind
crystallographic studies. Beamlines dedicated to biological SAXS that X-rays provide a more powerful and demanding tool than
have been constructed and help to consolidate the complementar- standard biophysical equipment. Therefore synchrotrons should
ity of SAXS and crystallography (Putnam et al., 2007). More re- not be employed as analytical tools to characterize unknown, per-
cently, the NMR community has embraced SAXS as a technique haps poorly behaving samples, and preliminary characterization by
that is not only complementary to high resolution solution struc- light scattering, gel-filtration or analytical ultracentrifugation is
ture analysis but that can be incorporated directly in structure recommended.
determination (Gabel et al., 2008; Grishaev et al., 2005). The many novel and exciting biological questions brought by
In addition to model validation (discussed in Section 4), SAXS new users of the technique require that laboratories specializing
can be used in crystallography as a tool for molecular replacement. in SAXS continue to push the boundaries in methods development.
The program FSEARCH (Hao, 2006) uses ab initio shape envelopes A number of research groups from around the world are actively
and bead or dummy residue models from EM or SAXS for the deter- involved in the development of advanced computational methods
mination of low-resolution phases. Following correct positioning of for the characterization of biomolecules using SAXS. The tools
the molecular envelope or bead/dummy model within the unit cell made available by these groups have had a significant impact on
the low-resolution phases are extended to crystallographic resolu- the field of structural biology, with automation of data collection,
tion. This promising method has been used successfully for several data reduction and analysis in particular making SAXS more acces-
proteins (Kollman and Quispe, 2005; Liu et al., 2003) and continues sible to the non-expert (Petoukhov et al., 2007; Round et al., 2008).
to be developed (Hong and Hao, 2009). The availability of beamlines for biological SAXS has also improved
A major obstacle in the determination of large (>30 kDa) pro- over the last few years, with the high-brilliance beamlines BL45XU
teins and complexes by NMR spectroscopy is the increased diffi- (Spring-8, Japan), ID14–3 (ESRF, France), and SAXS/WAXS (Austra-
culty of extracting useful structural information as the size of the lian Synchrotron, Australia) now in operation, with BioSAXS
system to be studied increases. As resonance overlap becomes a (PETRA III, Hamburg) expected to be operational in 2011. The
significant problem with the increased size of macromolecules, beamlines X33 (DORIS, Hamburg) (Roessle et al., 2007; Round
unambiguous sequence specific assignment of both the backbone et al., 2008), SYBILS (ALS, Berkley) (Hura et al., 2009), BL4–2 (SSRL,
and side-chains becomes difficult. This, combined with the disap- USA) and SWING (Soleil, Orsay) (David and Perez, 2009) offer auto-
pearance of peaks due to relaxation processes leads to a reduction mated sample changers, the latter one also combined with on-line
in the number of distance constraints typically used for structure HPLC purification and a UV–vis absorption monitoring system.
calculation. Orientation constraints derived from residual dipolar Other complementary techniques are also employed on-line, e.g.
couplings help to overcome the size limitations by providing resonant Raman spectroscopy at the ESRF ID-13 beamline (Davies
long-range information on the relative orientation of distant parts et al., 2009).
of the structure (Bax et al., 2001; Tjandra et al., 1997; Tolman et al., It should be mentioned that for many biological SAXS applica-
1995). However, they do not provide translational information and tions the use of modern laboratory instruments (for example those
by themselves cannot be used to generate an unambiguous solu- built by Bruker, Rigaku, Anton Paar and Hecus) permit one to col-
tion. SAXS data, providing information on the global shape of the lect data of sufficient quality. The laboratory SAXS experiment
macromolecule can be introduced into the structure calculation takes hours instead of seconds or minutes at a synchrotron, but
and reduce this ambiguity. The inclusion of potentials for the still provides data for computation of overall parameters, ab initio
refinement of NMR structures against SAXS data have been intro- shapes and rigid body analysis leading in some cases to exciting re-
duced into several popular structure calculation packages, for sults (Cramer et al., 2010; Hamley et al., 2010).
example the programs Xplor-NIH (Schwieters et al., 2003) and The programs mentioned in this review belonging to the ATSAS
CNS (Brunger, 2007; Brunger et al., 1999; Gabel et al., 2008; suite (Konarev et al., 2006; Petoukhov et al., 2007) are publicly
Grishaev et al., 2005), and have been shown to significantly available for download by academic users and for on-line access
improve the accuracy of calculated structures (Grishaev et al., from the EMBL-Hamburg web-site: http://www.embl-hamburg.
2005, 2008a). These methods have also been shown to be applica- de/ExternalInfo/Research/Sax/software.html/.
Fig. 9. A summary of the standard and more advanced tools for characterization of protein samples using SAXS. For detailed descriptions of the tools refer to the text. Several
suggested programs from the ATSAS package are indicated in capital letters.
In the present review we largely mentioned the SAXS applica- Bernadó, P., Mylonas, E., Petoukhov, M.V., Blackledge, M., Svergun, D.I., 2007.
Structural characterization of flexible proteins using small-angle X-ray
tions, which emerged from the collaborative projects at the EMBL
scattering. J. Am. Chem. Soc. 129, 5656–5664.
X33 beamline, but many extremely interesting SAXS applications Bernadó, P., Pérez, Y., Blobel, J., Fernández-Recio, J., Svergun, D.I., Pons, M., 2009.
are now being published by numerous groups worldwide (Fetler Structural characterization of unphosphorylated STAT5a oligomerization
et al., 2007; Hoiberg-Nielsen et al., 2009; West et al., 2008; equilibrium in solution by small-angle X-ray scattering. Protein Sci. 18, 716–
726.
Whitten et al., 2009). Especially powerful is the use of SAXS to- Bertini, I., Fragai, M., Luchinat, C., Melikian, M., Mylonas, E., Sarti, N., Svergun, D.I.,
gether with other structural and biochemical techniques, in a 2009. Interdomain flexibility in full-length matrix metalloproteinase-1 (MMP-
streamlined approach to characterize the structure and dynamical 1). J. Biol. Chem. 284, 12821–12828.
Blobel, J., Bernadó, P., Svergun, D.I., Tauler, R., Pons, M., 2009. Low-resolution
properties of proteins and protein complexes in solution. structures of transient protein–protein complexes using small-angle X-ray
scattering. J. Am. Chem. Soc. 131, 4378–4386.
Boehm, M.K., Woof, J.M., Kerr, M.A., Perkins, S.J., 1999. The fab and fc fragments of IgA1
exhibit a different arrangement from that in IgG: a study by X-ray and neutron
Acknowledgments solution scattering and homology modelling. J. Mol. Biol. 286, 1421–1447.
Bonnete, F., Finet, S., Tardieu, A., 1999. Second virial coefficient: variations with
The authors thank their collaborators and co-workers, in partic- lysozyme crystallization conditions. J. Cryst. Growth 196, 403–414.
Brunger, A.T., 2007. Version 1.2 of the crystallography and NMR system. Nat. Protoc.
ular at the EMBL (Hamburg): D. Franke, M. Gajda, C. Gorba, A. Kikh- 2, 2728–2733.
ney, P. Konarev, M. Petoukhov, M Roessle, W. Shang and Brunger, A.T., Adams, P.D., Rice, L.M., 1999. Annealing in crystallography: a powerful
A. Shkumatau for many stimulating discussions and critical com- optimization tool. Prog. Biophys. Mol. Biol. 72, 135–155.
Cammarata, M., Levantino, M., Schotte, F., Anfinrud, P.A., Ewald, F., Choi, J., Cupane,
ments. The authors acknowledge financial support from the HFSP
A., Wulff, M., Ihee, H., 2008. Tracking the structural dynamics of proteins in
Grant RGP0055/2006-C. H.D.T.M is supported by a fellowship from solution using time-resolved wide-angle X-ray scattering. Nat. Methods 5, 881–
the EMBL Interdisciplinary Postdocs programme (EIPOD). 886.
Chacon, P., Diaz, J.F., Moran, F., Andreu, J.M., 2000. Reconstruction of protein form
with X-ray solution scattering and a genetic algorithm. J. Mol. Biol. 299, 1289–
1302.
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Structural Characterization of Proteins and Complexes Using Small-Angle X-Ray Solution Scattering

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Journal of Structural Biology 172 (2010) 128–141

Contents lists available at ScienceDirect

Journal of Structural Biology

Structural characterization of proteins and complexes using small-angle

1. Introduction Production of good quality samples is a prerequisite for a suc-

cently, automated procedures have become available. In particular,

scattering curves can only be ﬁt within a restricted range (typically

assembly process), a model-independent estimate of K can be

Another important application of SAXS to rapidly characterize

Several methods have been developed to help simplify the anal-

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