Computational Molecular Biology

Biochem 218 – BioMedical Informatics 231

http://biochem218.stanford.edu/

Protein Structural Motifs

Doug Brutlag
Professor Emeritus
Biochemistry & Medicine (by courtesy)
 Homework 5: Phylogenies

• For this homework assignment take 20 to 30 protein sequences which

are at least 30% similar or better and:
o 1) make a multiple sequence alignment with them using ClustalW and
o 2) make two phylogenies, one using UPGMA method and the other using the
Neighbor Joining method
• Describe the resulting alignments and include graphic images of the
phylogenies in a message to [email protected]
• Mention if the trees seem reasonable biologically or taxonomically by
comparison with standard taxonomies
• Do the two trees have the same topology?
• Do the trees have the same branch lengths?
• If the two trees do not have the same topology or branch lengths,
describe the differences and indicate why you think the two trees
differ. Are the differences significant?
• Do the trees show evidence of paralogous evolution? Which nodes
are orthologous and which are paralogous bifurcations?
• Do the trees show evidence of either gene conversion or horizontal
gene transfer?
 Final Projects Due March 12

• Examples of Previous Final Projects

o http://biochem218.stanford.edu/Projects.html
• Critical review of any area of computational molecular biology.
o Area from the lectures but in more depth
o Any other area of bioinformatics or genomics focused on
computational approaches
• Proposed improvement or novel approach
• Can be a combined experimental/computational method.
• Could be an implementation or just pseudocode.
• Please do a MeSH literature search for Reviews on your topic.
Some useful MeSH terms include:
o Algorithms
o Statistics
o Molecular Sequence Data
o Molecular Structure etc.
• Please send a proposed final project topic to
[email protected] by next Friday
 Protein Structure Computational Goals

• Compare all known structures to each other

• Compute distances between protein structures
• Classify and organize all structures in a biologically
meaningful way
• Discover conserved substructure domain
• Discover conserved substructural motifs
• Find common folding patterns and structural/functional
motifs
• Discover relationship between structure and function.
• Study interactions between proteins and other proteins,
ligands and DNA (Protein Docking)
• Use known structures and folds to infer structure from
sequence (Protein Threading)
• Use known structural motifs to infer function from structure
• Many more…
 Structural Classification of Proteins (SCOP)
http://scop.berkeley.edu/

• Class
o Similar secondary
structure content
o All α, all β, alternating α/
β etc

• Fold (Architecture)
o Major structural similarity
o SSE’s in similar
arrangement

• Superfamily (Topology)
o Probable common
ancestry
o HMM family membership

• Family
o Clear evolutionary
relationship
o Pairwise sequence
similarity > 25%
 Classes of Protein Structures
• Mainly α
• Mainly β
 α β alternating
o Parallel β sheets, β-α-β
units

• α β
o Anti-parallel β sheets,
segregated α and β regions
o helices mostly on one side of
sheet
 Classes of Protein Structures

• Others
o Multi-domain, membrane and cell surface,
small proteins, peptides and fragments,
designed proteins
 Folds / Architectures
• Mainly α
• α/ βand α+β
o Bundle
o • Closed
Non-Bundle
• Mainly β • Barrel
o Single sheet • Roll, ...
o Roll • Open
o Barrel • Sandwich
o Clam • Clam, ...
o Sandwich
o Prism
o 4/6/7/8 Propeller
o Solenoid
The TIM Barrel Fold
A Conceptual Problem ...
Fold versus Topology

Another example:
Globin
vs.
Colicin
 PDB Protein Database
http://www.rcsb.org/pdb/

• Protein DataBase
o Multiple Structure Viewers
o Sequence & Structure Comparison Tools
o Derived Data
 SCOP
 CATH
 pFAM
 Go Terms
o Education on Protein Structure
o Download Structures and Entire Database
PDB Protein Database
http://www.rcsb.org/pdb/
PDB Protein Database
http://www.rcsb.org/pdb/
PDB Advanced Search for UniProt Entry
http://www.rcsb.org/pdb/
PDB Search Results
http://www.rcsb.org/pdb/
 PDB E. coli Hu Entry
http://www.rcsb.org/pdb/explore/explore.do?structureId=2O97
 PDB SimpleViewer
http://www.rcsb.org/pdb/
PDB Protein Workshop View
http://www.rcsb.org/pdb/
 PDB Derived Data
http://www.rcsb.org/pdb/
 Molecule of the Month: Enhanceosome
http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/current_month.html
 NCBI Structure Database
http://www.ncbi.nlm.nih.gov/Structure/

• Macromolecular Structures
• Related Structures
• View Aligned Structures & Sequences
• Cn3D: Downloadable Structure & Sequence Viewer
• CDD: Conserved Domain Database
o CD-Search: Protein Sequence Queries
o CD-TREE: Protein Classification Downloadable Application
o CDART: Conserved Domain Architecture Tool
• PubChem: Small Molecules and Biological Activity
• Biological Systems: BioCyc, KEGG and Reactome Pathways
• MMDB: Molecular Modeling Database
• CBLAST: BLAST sequence against PDB and Related Structure
Database
• IBIS: Inferred Biomolecular Interaction Server
• VAST Search: Structure Alignment Tool
 NCBI Structure Database
http://www.ncbi.nlm.nih.gov/Structure/
 NCBI Structure Database
http://www.ncbi.nlm.nih.gov/Structure/
 NCBI Cn3D Viewer
http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml
PyMol PDB Structure Viewer
http://www.pymol.org/
 Databases of Protein Folds

• SCOP (http://scop.berkeley.edu/)
o Structural Classification of Proteins
o Class-Fold-Superfamily-Family
o Manual assembly by inspection
• Superfamily (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/)
o HMM models for each SCOP fold
o Fold assignments to all genome ORFs
o Assessment of specificity/sensitivity of structure prediction
o Search by sequence, genome and keywords
• CATH (http://www.biochem.ucl.ac.uk/bsm/cath/)
o Class - Architecture - Topology - Homologous Superfamily
o Manual classification at Architecture level
o Automated topology classification using SSAP (Orengo & Taylor)
• FSSP (http://www2.embl-ebi.ac.uk/dali/fssp/ )
o Fully automated using the DALI algorithm (Holm & Sander)
o No internal node annotations
o Structural similarity search using DALI
SCOP Database of Protein Folds
http://scop.berkeley.edu/
 SCOP Hierarchy
http://scop.berkeley.edu/data/scop.b.html
 SCOP Alpha and Beta Proteins
http://scop.berkeley.edu/data/scop.b.d.html
 SCOP TIM Barrels
http://scop.berkeley.edu/data/scop.b.d.b.html
 SCOP Thiamin Phosphate Synthase
http://scop.berkeley.edu/data/scop.b.d.b.d.A.html
SCOP Thiamin Phosphate Synthase Entry
http://scop.berkeley.edu/
 SuperFamily HMM Fold Library
http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/
 SuperFamily Major Features
http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/
Genome Assignments by Superfamily
http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/
 Databases of Protein Folds

• SCOP (http://scop.berkeley.edu/)
o Structural Classification of Proteins
o Class-Fold-Superfamily-Family
o Manual assembly by inspection
• Superfamily (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/)
o HMM models for each SCOP fold
o Fold assignments to all genome ORFs
o Assessment of specificity/sensitivity of structure prediction
o Search by sequence, genome and keywords
• CATH (http://www.biochem.ucl.ac.uk/bsm/cath/)
o Class - Architecture - Topology - Homologous Superfamily
o Manual classification at Architecture level
o Automated topology classification using SSAP (Orengo & Taylor)
• FSSP (http://www2.embl-ebi.ac.uk/dali/fssp/ )
o Fully automated using the DALI algorithm (Holm & Sander)
o No internal node annotations
o Structural similarity search using DALI
CATH Protein Structure Classification
http://www.biochem.ucl.ac.uk/bsm/cath/
CATH Protein Structure Hierarchy
http://www.biochem.ucl.ac.uk/bsm/cath/
 CATH Protein Class Level
http://www.biochem.ucl.ac.uk/bsm/cath/
 CATH Orthogonal Bundle
http://www.biochem.ucl.ac.uk/bsm/cath/
 CATH Protein Summary
http://www.biochem.ucl.ac.uk/bsm/cath/
 CATH Protein Summary
http://www.biochem.ucl.ac.uk/bsm/cath/
 Databases of Protein Folds

• SCOP (http://scop.berkeley.edu/)
o Structural Classification of Proteins
o Class-Fold-Superfamily-Family
o Manual assembly by inspection
• Superfamily (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/)
o HMM models for each SCOP fold
o Fold assignments to all genome ORFs
o Assessment of specificity/sensitivity of structure prediction
o Search by sequence, genome and keywords
• CATH (http://www.biochem.ucl.ac.uk/bsm/cath/)
o Class - Architecture - Topology - Homologous Superfamily
o Manual classification at Architecture level
o Automated topology classification using SSAP (Orengo & Taylor)
• FSSP (http://www2.embl-ebi.ac.uk/dali/fssp/ )
o Fully automated using the DALI algorithm (Holm & Sander)
o No internal node annotations
o Structural similarity search using DALI
 FSSP Database
http://srs.ebi.ac.uk/srsbin/cgi-bin/wgetz?-page+LibInfo+-lib+FSSP
 Dali Server
http://www.ebi.ac.uk/dali/
 DALI Database (Liisa Holm)
http://ekhidna.biocenter.helsinki.fi/dali/start
 Protein Fold Prediction: Swiss Model
http://swissmodel.expasy.org/

• Amos Bairoch, Swiss Bioinformatics Institute, SBI

• Threading and Template Discovery
• Workspace for saving Template Results
• Domain Annotation
• Structure Assessment
• Template Library
• Structures & Models
• Documentation and Tutorials
Protein Fold Prediction: Swiss Model
http://swissmodel.expasy.org/
Automatic Protein Fold Prediction
http://swissmodel.expasy.org/
Automatic Protein Fold Prediction Results
http://swissmodel.expasy.org/
Automatic Protein Fold Prediction Results
http://swissmodel.expasy.org/
Automatic Protein Fold Prediction Results
http://swissmodel.expasy.org/
 Protein Fold Prediction: phyre
http://www.sbg.bio.ic.ac.uk/~phyre/

• Michael Sternberg, Structural Bioinformatics Group,

Imperial College London
• Protein structure prediction on the web: a case study
using the Phyre server Kelley LA and Sternberg MJE.
Nature Protocols 4, 363 - 371 (2009)
• Protein Homology/analogY Recognition Engine
Protein Fold Prediction: phyre
http://www.sbg.bio.ic.ac.uk/~phyre/
Protein Fold Prediction: phyre
http://www.sbg.bio.ic.ac.uk/~phyre/
Protein Fold Prediction: phyre
http://www.sbg.bio.ic.ac.uk/~phyre/
 Protein Fold Prediction: PsiPred
http://bioinf4.cs.ucl.ac.uk:3000/psipred/

• Kevin Bryson and David Jones, University College

London
• Predicts Secondary Structure of single molecules
• Predicts Transmembrane Topology
• Three Fold Recognition methods
Protein Fold Prediction: PsiPred
http://bioinf4.cs.ucl.ac.uk:3000/psipred/
Protein Fold Prediction: PsiPred
http://bioinf4.cs.ucl.ac.uk:3000/psipred/
 Protein Fold Prediction: Predict Protein
http://www.predictprotein.org/

• Burkhard Rost, Columbia

• Methods
o MaxHom : multiple alignment
o PSI-BLAST : iterated profile
o searchProSite : functional motifs
o SEG : composition-bias
o ProDom : domain assignment
o PredictNLS : nuclear localisation signal
o PHDsec : secondary structure
o PHDacc : solvent accessibility
o Globe : globularity of proteins
o PHDhtm : transmembrane helices
o PROFsec : secondary structure
o PROFacc : solvent accessibilityCoils : coiled-coil regions
o CYSPRED : cysteine bridges
o Topits : fold recognition by threading
Protein Fold Prediction: Predict Protein
http://www.predictprotein.org/
 Automating Structure Classification,
Fold & Function Detection

• Growth of PDB demands automated

techniques for classification and fold detection
• Protein Structure Comparison
o computing structure similarity based on metrics
(distances)
o identifying protein function
o understanding functional mechanism
o identifying structurally conserved regions in the
protein
o finding binding sites or other functionally
important regions of the protein
 Structure Superposition

• Find the transformation matrix that best overlaps the table

and the chair
• i.e. Find the transformation matrix that minimizes the root
mean square deviation between corresponding points of
the table and the chair
• Correspondences:
o Top of chair to top of table
o Front of chair to front of table, etc.
 Absolute Orientation Algorithm
http://www-mtl.mit.edu/researchgroups/itrc/ITRC_publication/horn_publications.html

Closed-form solution of absolute orientation using unit quate

Berthold K.P. Horn, J.Opt.Soc.Am,

+ April 1987, Vol 4, No. 4

The key is finding corresponding points between

the two structures
 Algorithms for Structure Superposition
• Distance based methods:
 DALI (Holm & Sander): Aligning scalar distance plots

STRUCTAL (Gerstein & Levitt): Dynamic programming
using pair-wise inter-molecular distances

SSAP (Orengo & Taylor): Dynamic programming using
intra-molecular vector distances

MINAREA (Falicov and Cohen): Minimizing soap-bubble
surface area
 CE (Shindyalov & Bourne)
• Vector based methods:

VAST (Bryant): Graph theory based secondary structure
alignment

3D Search (Singh and Brutlag) & 3D Lookup (Holm
and Sander): Fast secondary structure index lookup
• Both

LOCK (Singh & Brutlag) LOCK2 (Ebert & Brutlag):
Hierarchically uses both secondary structure vectors and
atomic distances
 DALI

An intra-molecular distance plot for myoglobin

 DALI

• Based on aligning 2-D intra-molecular distance

matrices
• Computes the best subset of corresponding
residues from the two proteins such that the
similarity between the 2-D distance matrices is
maximized
• Searches through all possible alignments of
residues using Monte-Carlo and Branch-and-
Bound algorithms

Score(i, j) = 1.5 - |distanceA(i, j) - distanceB(i, j)|

 STRUCTAL
• Based on Iterative Dynamic Programming to
align inter-molecular distances
• Pair-wise alignment score in each square of
the matrix is inversely proportional to
distance between the two atoms

12 3 4 5 6 1 2 3 4 5 6
1 1
2 2
3 3
4 4
5 5
6 6
 VAST - Vector Alignment Search Tool
• Aligns only secondary structure elements (SSE)

• Represents each SSE as a vector

• Finds all possible pairs of vectors from the two structures that are
similar

• Uses a graph theory algorithm to find maximal subset of similar

vector pairs

• Overall alignment score is based on the number of similar pairs of

vectors between the two structures
 Algorithms for Structure Superposition
• Atomic distance based methods:
 DALI (Holm and Sander): Aligning scalar distance plots
 STRUCTAL (Gerstein and Levitt): Dynamic programming
using pair wise inter-molecular distances
 SSAP (Orengo and Taylor): Dynamic programming using
intra-molecular vector distances
 MINAREA (Falicov and Cohen): Minimizing soap-bubble
surface area
• Vector based methods:
 VAST (Bryant): Graph theory based secondary structure
alignment
 3dSearch (Singh and Brutlag): Fast secondary structure index
lookup
• Use both SSE vectors and atomic distances

LOCK (Singh and Brutlag): Hierarchically uses both
secondary structure vectors and atomic distances
LOCK - Creating Secondary Structure Vectors
 Comparing Secondary Structure Vectors

θ Orientation Independent Scores:

i k
S = S(|angle θ(i,k) - angle φ(p,r)|)
S = S(|distance(i,k) - distance(p,r)|)
S = S(|length(i) - length(p)|)+
φ S(|length(p) - length(r)|)
p
r Orientation Dependent Scores:
S = S(angle(k,r))
S = S(distance(k,r))

M
2M
2 - M d
S(d) = d
1+ d0
d0
-M
Aligning Secondary Structure Vectors

H H S S
S Best local alignment :
H HHSS
S SHSSH
S
H
 Three Step Algorithm

• Local Secondary Structure Superposition

o Find an initial superposition of the two proteins by using
dynamic programming to align the secondary structure
vectors

• Atomic Superposition
o Apply a greedy nearest neighbor method to minimize the
RMSD between the C-α atoms from query and the target
(i.e. find the nearest local minimum in the alignment
space)

• Core Superposition
o Find the best sequential core of aligned C-α atoms and
minimize the RMSD between them
Step 1: Local Secondary Structure Superposition

S4 H3
S4
S2
H1 H1
H3
S2
Step 1: Local Secondary Structure Superposition

B3
A4 B4
A2
A1 B1
A3 B2

pair # of aligned vectors total alignment score

A1,A2 2 32
B2,B3
A3,A4
3 71
B3,B4
Step 1: Local Secondary Structure Superposition
Step 2: Atomic Superposition
Step 3: Core Superposition
LOCK 2: Secondary Structure Element
Alignment

φ
ψ Ф
d

Superimpose vectors and

θ Compare internal distances in
Represent
score
Restore
alignment
secondary
secondary
using
structure
both
ψ order to find equivalent
φ structure
orientation
element representation
elements
independent
as vectors
and
secondary structure elements
d orientation dependent scores
 Residue Alignment

EEKSAVTALWGKV--
GDKKAINKIWPKIYK

superposition residue registration

• Naïve approach:
Nearest neighbor alpha
carbons
 Beta Carbons Encode Directional
Information

θ = Angle between Cα and

Cβ vectors
d = distance between Cβ atom
(maximum 6Ǻ)
New Residue Alignment

Cβ
 Improvements in Consistency
• Consistency: measures the adherence to the transitivity property
among all triples of protein structures in a given superfamily

Globin Immunoglobulin
Superfa Superfamily
mily

Alpha carbon 74.3% 58.6%

distances

Beta carbon 80% 59.9%

positions

% increase in 37.0% 77.8%

aligned residues

(less than 10% pairwise sequence identity)

 New LOCK 2 Properties

• Changes to secondary structure element alignment

phase allow for recognition of more distant structural
relationships
• Metric scoring function:
1-score(A,B) + 1-score(B,C) ≤ 1-score(A,C)
• Biologically relevant residue alignment
• Highly consistent alignments
• Symmetric
• Assessment of statistical significance
 FoldMiner: Structure Similarity Search
Based on LOCK2 Alignment

• FoldMiner aligns query structure with all

database structures using LOCK2

• FoldMiner up weights secondary structure

elements in query that are aligned more often

• FoldMiner outperforms CE and VAST is

searches for structure similarity
 Receiver-Operating Characteristic (ROC) Curves

Ra Fold 16
14
nk 12
10
1 Immunoglobuli 8
6
n Positives
4
Number
2 of True
2 Immunoglobuli 0
0 5 10 15
n
...

...

Number of False Positives

3 p53
• Gold standard: Structural Classification of Proteins
(SCOP)
o SCOP folds: similar arrangement and connectivity of
secondary structure elements
 Comparing ROC Curves

40
Number of True Positives

35 • Area under the ROC

30
25 curve correlates with
20 the property of ranking
15
10
CE
VAST
true positives ahead of
5 FoldMiner false positives
0
0 100 200 300
• Curves may terminate
25 at different numbers of
20 true and false positives
15 • Areas can only be
10 VAST
directly compared if
5 CE calculated at points
FoldMiner where the two curves
0
0 5 10 15 20
cross over one another
Number of False Positives
Comprehensive Analysis of ROC Curves
 Motif Alignment Results

Families Superfamilies

eMOTIFs 96.4% 91.6%

Prosite patterns 97.4% 92.6%

LOCK2 Superposition Web Site
http://brutlag.stanford.edu/lock2/
LOCK2 Superposition Web Site
http://brutlag.stanford.edu/lock2/
PyMol Display of LOCK2 Superposition
FoldMiner Structure Search
http://brutlag.stanford.edu/foldminer/
FoldMiner Myoglobin Structure Search
http://brutlag.stanford.edu/foldminer/
FoldMiner Myoglobin Structure Search
http://brutlag.stanford.edu/foldminer/
FoldMiner Myoglobin Structure Search
http://brutlag.stanford.edu/foldminer/
FoldMiner Myoglobin Structure Search
http://brutlag.stanford.edu/foldminer/
 ModLink+
http://sbi.imim.es/modlink/