3D Structure Prediction

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3D Structure Prediction Assessment Pt. 2

• David Wishart
• Rm. 2123 Dent/Pharm Centre
• david.wishart_at_ualberta.ca

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3D Structure Generation

• X-ray Crystallography
• NMR Spectroscopy
• Homology or Comparative Modelling
• Threading (1D and 2D threading)
• Secondary Structure Prediction
• Ab initio Structure Prediction

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Outline

• Threading (1D and 3D threading)
• Secondary Structure Prediction
• Ab initio Structure Prediction
• Structure Evaluation Assessment
• PERL and PDB

4
Definition

• Threading - A protein fold recognition technique
  that involves incrementally replacing the
  sequence of a known protein structure with a
  query sequence of unknown structure. The new
  model structure is evaluated using a simple
  heuristic measure of protein fold quality. The
  process is repeated against all known 3D
  structures until an optimal fit is found.

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Why Threading?

• Secondary structure is more conserved than
  primary structure
• Tertiary structure is more conserved than
  secondary structure
• Therefore very remote relationships can be better
  detected through 2o or 3o structural homology
  instead of sequence homology

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Visualizing Threading
THREADINGSEQNCEECNQESGNI ERHTHREADINGSEQNCETHREAD
GSEQNCEQCQESGIDAERTHR...
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Visualizing Threading
THREADINGSEQNCEECNQESGNI ERHTHREADINGSEQNCETHREAD
GSEQNCEQCQESGIDAERTHR...
T
H
R
E
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Visualizing Threading
THREADINGSEQNCEECNQESGNI ERHTHREADINGSEQNCETHREAD
GSEQNCEQCQESGIDAERTHR...
T
H
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Visualizing Threading
THREADINGSEQNCEECNQESGNI ERHTHREADINGSEQNCETHREAD
GSEQNCEQCQESGIDAERTHR...
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Visualizing Threading
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Threading

• Database of 3D structures and sequences
• Protein Data Bank (or non-redundant subset)
• Query sequence
• Sequence lt 25 identity to known structures
• Alignment protocol
• Dynamic programming
• Evaluation protocol
• Distance-based potential or secondary structure
• Ranking protocol

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2 Kinds of Threading

• 2D Threading or Prediction Based Methods (PBM)
• Predict secondary structure (SS) or ASA of query
• Evaluate on basis of SS and/or ASA matches
• 3D Threading or Distance Based Methods (DBM)
• Create a 3D model of the structure
• Evaluate using a distance-based hydrophobicity
  or pseudo-thermodynamic potential

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2D Threading Algorithm

• Convert PDB to a database containing sequence, SS
  and ASA information
• Predict the SS and ASA for the query sequence
  using a high-end algorithm
• Perform a dynamic programming alignment using the
  query against the database (include sequence, SS
  ASA)
• Rank the alignments and select the most probable
  fold

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Database Conversion
gtProtein1 THREADINGSEQNCEECNQESGNI HHHHHHCCCCEEEEE
CCCHHHHHH ERHTHREADINGSEQNCETHREAD HHCCEEEEECCCCCH
HHHHHHHHH
gtProtein2 QWETRYEWQEDFSHAECNQESGNI EEEEECCCCHHHHHH
HHHHHHHHH YTREWQHGFDSASQWETRA CCCCEEEEECCCEEEEECC
gtProtein3 LKHGMNSNWEDFSHAECNQESG EEECCEEEECCCEEECC
CCCCC
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Secondary Structure
-
-
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2o Structure Identification

• DSSP - Database of Secondary Structures for
  Proteins (swift.embl-heidelberg.de/dssp)
• VADAR - Volume Area Dihedral Angle Reporter
  (redpoll.pharmacy.ualberta.ca)
• PDB - Protein Data Bank (www.rcsb.org)

QHTAWCLTSEQHTAAVIWDCETPGKQNGAYQEDCA HHHHHHCCEEEEEE
EEEEECCHHHHHHHCCCCCCC
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Accessible Surface Area
Reentrant Surface
Accessible Surface
Solvent Probe
Van der Waals Surface
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ASA Calculation

• DSSP - Database of Secondary Structures for
  Proteins (swift.embl-heidelberg.de/dssp)
• VADAR - Volume Area Dihedral Angle Reporter
  (www.pence.ualberta.ca/ftp/vadar)
• GetArea - www.scsb.utmb.edu/getarea/area_form.html
  

QHTAWCLTSEQHTAAVIWDCETPGKQNGAYQEDCAMD
BBPPBEEEEEPBPBPBPBBPEEEPBPEPEEEEEEEEE 10562987994
15251510478941496989999999
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Other ASA sites

• Connolly Molecular Surface Home Page
• http//www.biohedron.com/
• Naccess Home Page
• http//sjh.bi.umist.ac.uk/naccess.html
• ASA Parallelization
• http//cmag.cit.nih.gov/Asa.htm
• Protein Structure Database
• http//www.psc.edu/biomed/pages/research/PSdb/

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2D Threading Algorithm

• Convert PDB to a database containing sequence, SS
  and ASA information
• Predict the SS and ASA for the query sequence
  using a high-end algorithm
• Perform a dynamic programming alignment using the
  query against the database (include sequence, SS
  ASA)
• Rank the alignments and select the most probable
  fold

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2o Structure Prediction

• Statistical (Chou-Fasman, GOR)
• Homology or Nearest Neighbor (Levin)
• Physico-Chemical (Lim, Eisenberg)
• Pattern Matching (Cohen, Rooman)
• Neural Nets (Qian Sejnowski, Karplus)
• Evolutionary Methods (Barton, Niemann)
• Combined Approaches (Rost, Levin, Argos)

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Chou-Fasman Statistics
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The PhD Approach
PRFILE...
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The PhD Algorithm

• Search the SWISS-PROT database and select high
  scoring homologues
• Create a sequence profile from the resulting
  multiple alignment
• Include global sequence info in the profile
• Input the profile into a trained two-layer neural
  network to predict the structure and to
  clean-up the prediction

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Prediction Performance
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Best of the Best

• PredictProtein-PHD (72)
• http//cubic.bioc.columbia.edu/predictprotein
• Jpred (73-75)
• http//jura.ebi.ac.uk8888/
• PREDATOR (75)
• http//www.embl-heidelberg.de/cgi/predator_serv.pl
  
• PSIpred (77)
• http//insulin.brunel.ac.uk/psipred

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ASA Prediction

• PredictProtein-PHDacc (58)
• http//cubic.bioc.columbia.edu/predictprotein
• PredAcc (70?)
• condor.urbb.jussieu.fr/PredAccCfg.html

QHTAW...
QHTAWCLTSEQHTAAVIW BBPPBEEEEEPBPBPBPB
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2D Threading Algorithm

• Convert PDB to a database containing sequence, SS
  and ASA information
• Predict the SS and ASA for the query sequence
  using a high-end algorithm
• Perform a dynamic programming alignment using the
  query against the database (include sequence, SS
  ASA)
• Rank the alignments and select the most probable
  fold

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Dynamic Programming
G
E
N
E
T
I
C
S
G
60
40
30
20
20
0
10
0
E
40
50
30
30
20
0
10
0
N
30
30
40
20
20
0
10
0
E
20
20
20
30
20
10
10
0
S
20
20
20
20
20
0
10
10
I
10
10
10
10
10
20
10
0
S
0
0
0
0
0
0
0
10
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Sij (Identity Matrix)
A C D E F G H I K L M N P Q R S T V W Y A 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 0 1 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 D 0 0 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 E 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 F 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 G 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 H 0 0
0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 I 0 0 0 0 0 0
0 1 0 0 0 0 0 0 0 0 0 0 0 0 K 0 0 0 0 0 0 0 0 1 0
0 0 0 0 0 0 0 0 0 0 L 0 0 0 0 0 0 0 0 0 1 0 0 0 0
0 0 0 0 0 0 M 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
0 0 N 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 P 0
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 Q 0 0 0 0 0
0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 R 0 0 0 0 0 0 0 0 0
0 0 0 0 0 1 0 0 0 0 0 S 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 1 0 0 0 0 T 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0 0 0 V 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 W
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 Y 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
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A Simple Example...
A A T V D A 1 V V D
A A T V D A 1 1 V V D
A A T V D A 1 1 0 0 0 V V D
A A T V D A 1 1 0 0 0 V 0 V D
A A T V D A 1 1 0 0 0 V 0 1 1 V D
A A T V D A 1 1 0 0 0 V 0 1 1 2 V D
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A Simple Example...
A A T V D A 1 1 0 0 0 V 0 1 1 2 1 V D
A A T V D A 1 1 0 0 0 V 0 1 1 2 1 V 0 1
1 2 2 D 0 1 1 1 3
A A T V D A 1 1 0 0 0 V 0 1 1 2 1 V 0 1
1 2 2 D 0 1 1 1 3
A A T V D A - V V D
A A T V D A V V D
A A T V D A V - V D
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Lets Include 2o info ASA
H E C
E P B
Sij
Sij
H 1 0 0 E 0 1 0 C 0 0 1
E 1 0 0 P 0 1 0 B 0 0 1
strc
asa
Sij k1Sij k2Sij k3Sij
total
seq
strc
asa
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A Simple Example...
E E E C C
E E E C C
E E E C C
A A T V D A 2 V V D
A A T V D A 2 2 V V D
A A T V D A 2 2 1 0 0 V V D
E E C C
E E C C
E E C C
E E E C C
E E E C C
E E E C C
A A T V D A 2 2 1 0 0 V 1 V D
A A T V D A 2 2 1 0 0 V 1 3 3 V D
A A T V D A 2 2 1 0 0 V 1 3 3 3 V D
E E C C
E E C C
E E C C
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A Simple Example...
E E E C C
E E E C C
E E E C C
A A T V D A 2 2 1 0 0 V 1 3 3 3 2 V D
A A T V D A 2 2 1 0 0 V 1 3 3 3 2 V 0 2
3 5 4 D 0 2 3 4 7
A A T V D A 2 2 1 0 0 V 1 3 3 3 2 V 0 2
3 5 4 D 0 2 3 4 7
E E C C
E E C C
E E C C
A A T V D A - V V D
A A T V D A V V D
A A T V D A V - V D
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2D Threading Performance

• In test sets 2D threading methods can identify
  30-40 of proteins having very remote homologues
  (i.e. not detected by BLAST) using minimal
  non-redundant databases (lt700 proteins)
• If the database is expanded 4x the performance
  jumps to 70-75
• Performs best on true homologues as opposed to
  postulated analogues

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2D Threading Advantages

• Algorithm is easy to implement
• Algorithm is very fast (10x faster than 3D
  threading approaches)
• The 2D database is small (lt500 kbytes) compared
  to 3D database (gt1.5 Gbytes)
• Appears to be just as accurate as DBM or other 3D
  threading approaches
• Very amenable to web servers

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Servers - PredictProtein
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Servers - 123D
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Servers - GenThreader
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Servers - LIBRA 1
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More Servers - www.bronco.ualberta.ca
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2D Threading Disadvantages

• Reliability is not 100 making most threading
  predictions suspect unless experimental evidence
  can be used to support the conclusion
• Does not produce a 3D model at the end of the
  process
• Doesnt include all aspects of 2o and 3o
  structure features in prediction process
• PSI-BLAST may be just as good (faster too!)

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Making it Better

• Include 3D threading analysis as part of the 2D
  threading process -- offers another layer of
  information
• Include more information about the coil state
  (3-state prediction isnt good enough)
• Include other biochemical (ligands, function,
  binding partners, motifs) or phylogenetic
  (origin, species) information

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Outline

• Threading (1D and 3D threading)
• Secondary Structure Prediction
• Ab initio Structure Prediction
• Structure Evaluation Assessment
• PERL and PDB

46
Ab Initio Prediction

• Predicting the 3D structure without any prior
  knowledge
• Used when homology modelling or threading have
  failed (no homologues are evident)
• Equivalent to solving the Protein Folding
  Problem
• Still a research problem

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Polypeptides can be...

• Represented by a range of approaches or
  approximations including
• all atom representations in cartesian space
• all atom representations in dihedral space
• simplified atomic versions in dihedral space
• tube/cylinder/ribbon representations
• lattice models

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Ab Initio Folding

• Two Central Problems
• Sampling conformational space (10100)
• The energy minimum problem
• The Sampling Problem (Solutions)
• Lattice models, off-lattice models, simplified
  chain methods, parallelism
• The Energy Problem (Solutions)
• Threading energies, packing assessment, topology
  assessment

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A Simple 2D Lattice
3.5Å
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Lattice Folding
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Lattice Algorithm

• Build a n x m matrix (a 2D array)
• Choose an arbitrary point as your N terminal
  residue (start residue)
• Add or subtract 1 from the x or y position of
  the start residue
• Check to see if the new point (residue) is off
  the lattice or is already occupied
• Evaluate the energy
• Go to step 3) and repeat until done

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Lattice Energy Algorithm

• Red hydrophobic, Blue hydrophilic
• If Red is near empty space E E1
• If Blue is near empty space E E-1
• If Red is near another Red E E-1
• If Blue is near another Blue E E0
• If Blue is near Red E E0

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More Complex Lattices
54
3D Lattices
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Really Complex 3D Lattices
J. Skolnick
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Lattice Methods
Advantages
Disadvantages

• Easiest and quickest way to build a polypeptide
• Implicitly includes excluded volume
• More complex lattices allow reasonably accurate
  representation

• At best, only an approximation to the real thing
• Does not allow accurate constructs
• Complex lattices are as costly as the real thing

57
Non-Lattice Models
3.5 Å
H
R
Resi
C
H
1.53 Å
1.00 Å
1.32 Å
C
N
1.47 Å
1.24 Å
O
C
Resi1
H
R
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Vistraj Foldtraj

• Chris Hogue Howard Feldman (SLRI)
• Uses simplified Ca chain to represent polypeptide
  backbone
• Generates a simplified self-avoiding chain of
  100 residues in 3 sec
• Uses a binary tree search to look for potential
  collisions in 3D space
• Reconstructs full polypeptide from Cas

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Simplified Chain Representation
4
q
3
f
2
1
Spherical Coordinates
60
The Search Sphere
Helix
Coil
b-Sheet
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Building a Ca Peptide Chain
n 3 n 5 n 7 n 9
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Simplified Chain Representation
Reconstructing backbone atoms from Ca atoms
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Best Method So Far...
Rosetta - David Baker
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Blue Gene and Protein Folding
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Outline

• Threading (1D and 3D threading)
• Secondary Structure Prediction
• Ab initio Structure Prediction
• Structure Evaluation Assessment
• PERL and PDB

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Why Assess Structure?

• A structure can (and often does) have mistakes
• A poor structure will lead to poor models of
  mechanism or relationship
• Unusual parts of a structure may indicate
  something important (or an error)

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Famous bad structures

• Azobacter ferredoxin (wrong space group)
• Zn-metallothionein (mistraced chain)
• Alpha bungarotoxin (poor stereochemistry)
• Yeast enolase (mistraced chain)
• Ras P21 oncogene (mistraced chain)
• Gene V protein (poor stereochemistry)

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How to Assess Structure?

• Assess experimental fit (look at R factor or
  rmsd)
• Assess correctness of overall fold (look at
  disposition of hydrophobes)
• Assess structure quality (packing,
  stereochemistry, bad contacts, etc.)

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A Good Protein Structure..
X-ray structure NMR structure

• R 0.59 random chain
• R 0.45 initial structure
• R 0.35 getting there
• R 0.25 typical protein
• R 0.15 best case
• R 0.05 small molecule

• rmsd 4 Å random
• rmsd 2 Å initial fit
• rmsd 1.5 Å OK
• rmsd 0.8 Å typical
• rmsd 0.4 Å best case
• rmsd 0.2 Å dream on

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A Good Protein Structure..

• Minimizes disallowed torsion angles
• Maximizes number of hydrogen bonds
• Maximizes buried hydrophobic ASA
• Maximizes exposed hydrophilic ASA
• Minimizes interstitial cavities or spaces

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A Good Protein Structure..

• Minimizes number of bad contacts
• Minimizes number of buried charges
• Minimizes radius of gyration
• Minimizes covalent and noncovalent (van der Waals
  and coulombic) energies

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Radius Radius of Gyration

• RAD 3.875 x NUMRES 0.333 (Folded)
• RADG 0.41 x (110 x NUMRES) 0.5 (Unfolded)

Radius Radius of Gyration
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Packing Volume
Loose Packing Dense Packing Protein
Proteins are Densely Packed
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Accessible Surface Area
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Accessible Surface Area

• Solvation free energy is related to ASA
• DG SDsiAi
• Proteins typically have 60 of their ASA
  comprised of polar atoms or residues
• Proteins typically have 40 of their ASA
  comprised of nonpolar atoms or residues
• DASA (obs - exp.) reveals shape/roughness

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Structure Validation Servers

• WhatIf Web Server - http//www.cmbi.kun.nl1100/WI
  WWWI/
• Biotech Validation Suite - http//biotech.ebi.ac.u
  k8400/cgi-bin/sendquery
• Verify3D -
  http//www.doe-mbi.ucla.edu/Services/Verify_3D/
• VADAR - http//redpoll.pharmacy.ualberta.ca

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Structure Validation Programs

• PROCHECK - http//www.biochem.ucl.ac.uk/roman/pr
  ocheck/procheck.html
• PROSA II - http//lore.came.sbg.ac.at/People/mo/Pr
  osa/prosa.html
• VADAR - http//www.pence.ualberta.ca/ftp/vadar/
• DSSP - http//www.embl-heidelberg.de/dssp/

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Procheck
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Slides Located At...
http//redpoll.pharmacy.ualberta.ca