HOMOLOGY MODELLING

Chris Wilton
 Homology Modelling

§ What is it and why do we need it?

• principles of modelling, applications available

§ Using Swiss-Model
• preparation, workspace tools, automated-mode
• refining with manually-adjusted alignments

§ Model Assessment
• using SwissModel tools to check model quality
• what to look for, what we expect…
 What is Homology Modelling?
§ Given an unknown protein, make an informed
guess on its 3D structure based on its sequence:
• Search structure databases for homologous sequences
• Transfer coordinates of known protein onto unknown

MQEQLTDFSKVETNLISW-QGSLETVEQMEPWAGSDANSQTEAY
| |..|. ||| ... |..||.|.| | |||..|
MHQQVSDYAKVEHQWLYRVAGTIETLDNMSPANHSDAQTQAA

| = Identity
. | = Homology
 Why Modelling is Necessary

§ Current structure determination methods:

• NMR – conc. protein solution; limited size + resolution
• XRay Crystallography – protein crystals, high resolution
• Expensive, slow, difficult (especially membrane proteins!)

§ Can’t keep up with growth rate of sequence

databases:
• PDB: 40132 structures ( 14/11/2006 )
• pairsdb: 4000000+ sequences ( 11/2006 )
Current (Free) Servers & Software
§ SWISSMODEL (www)
http://swissmodel.expasy.org/SWISS-MODEL.html

§ 3D-Jigsaw (www)
http://www.bmm.icnet.uk/servers/3djigsaw/
§ ESyPred3D (www)
http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/
§ WHATIF (www)
http://swift.cmbi.kun.nl/WIWWWI
§ CPH Models 2.0 (www)
http://www.cbs.dtu.dk/services/CPHmodels
§ MODELLER 8v2 (standalone for windows, mac, linux; also web submission)
http://salilab.org/modeller
http://alto.compbio.ucsf.edu/modweb-cgi/main.cgi
 Assumptions & Principles
§ Increase in sequence identity correlates with
increase in structural similarity

§ RMSD of core α-carbon coordinates for two

homologous proteins sharing 50% identity
expected at ~1Å (GLY 3.5Å, helix 5Å diameter)

§ Theoretical models are low resolution, and

depend on quality of input alignment!
The SwissModel Workspace
 Template Search

Search structure databases for

potential homologues, and obtain
family information where possible.
 Preparation

§ Automated mode:
• Select a template structure with high % identity and
average resolution, or let SwissModel choose best one.

§ Alignment mode:
• Obtain multiple alignment between possible templates
and your query sequence.
• Check alignment – are key motifs correctly aligned?
Manually adjust as necessary, using Jalview.
• Upload alignment, select template, and submit.
Submit a Model Request

Automated Mode
Submit a Model Request

Alignment Mode
 Hidden Modelling Steps
§ Automated mode, multiple templates:
• Superimposition and optimisation of corresponding α-carbon
pairs for all template structures
• Alignment of all residues of template structures: acceptable
alpha-carbon atoms located within 3Å radius of mean

§ All modes:
• Framework construction of query protein structure, using
coordinates of template structure, or mean coordinate positions
if multiple templates.
• Building unconserved loop regions / insertions, closing gaps in
backbone - uses penta-peptide fragment library derived from all
PDB entries of resolution 2Å or better (note: this can fail badly!)
 Output
§ Within a few minutes of submission, your results
are returned to the Workspace.

§ Output Page:
1. Your model in pdb format (plus simple viewer)
2. Query to template alignment
3. Simple assessment graphs
4. Logging data of the modelling process

§ Save the model in DeepView Project format, then

open in DeepView, and colour by B-factor.
 Multiple Sequence Alignment

§ More information than pairwise alignment

§ Shows conserved regions in protein family

• Critical secondary structure elements

§ Shows strongly conserved residues and

motifs
• Structural importance
• Functional importance
 Alignment Adjustment

§ Look at alignment used to select template

• Check alignment against 3D structure (Jalview)
• Secondary structure elements preserved?
• Motifs and key residues aligned?
• Are gaps in acceptable locations (ie: loops)?

§ Try to improve alignment manually ...

§ Or use different alignment application
 The SwissModel Repository

• Contains theoretical models ( 996876 on 05/09/2006 )

• Searchable by UniProt / Trembl accession number
• Fully automated procedure - models may be bad!
• It can be found here:
http://swissmodel.expasy.org/repository

• ModBase also holds threshold-validated theoretical

models [ 4064704 structures currently ]:
http://alto.compbio.ucsf.edu/modbase-cgi/index.cgi
Model Assessment

Use SwissModel Workspace tools:

 Model Evaluation
§ Look at model in DeepView: is it globular and
compact, are there odd unstructured loops?

§ Colour by B-factor and note bad areas – in loops,

or in important secondary elements?

§ WhatCheck - always lots of errors! Look for

packing, inside-outside, torsion angle errors.

§ Check Solvation Profile – how well packed is the

model? Are there regions which are too loose,
and therefore solvent-exposed?
Torsion Angles
Ramachandran Plot

Experimentally Resolved Model

(NMR, XRay):
• Rare for residues other than
Glycine & Alanine to be in
disallowed regions

Homology Model:
• Bad residues common
• May have to remodel small
sections and loops
• Large residues in bad
regions worth further
investigation …
Solvation Profile

• Long stretches
above zero
probably loops
• Most-negative
regions well
buried in core
• Functional
residues above
zero!
• Majority of
residues must
be below zero
• Compare your
model to pdb
template!
 Further details

§ DeepView tutorial
– http://au.expasy.org/spdbv/text/tutorial.htm

§ Advanced tutorial on SwissModel & DeepView

– http://www.usm.maine.edu/~rhodes/SPVTut/text/HGHomMod.html
 Suggested Reading

§ Swiss-Model’s advice:
– http://www.expasy.org/swissmod/SM_ModelAccuracy.html

§ A good interactive tutorial on Protein Modelling:

– http://www.ncbi.nlm.nih.gov/Class/minicourses/x1a.html
References
Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-
MODEL: an automated protein homology-modeling server. Nucleic
Acids Research 31:3381-3385.

Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-

PdbViewer: An environment for comparative protein modelling.
Electrophoresis 18:2714-2723.

Peitsch, M. C. (1995) Protein modeling by Email. Bio/

Technology 13:658-660.

R.W.W.Hooft, G.Vriend, C.Sander and E.E.Abola, Errors in protein

structures. Nature 381, 272 (1996).

Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963). J

Mol Biol 7:95-99.
 Demonstrations
§ Uniprot entry Q11CR8_MESSB is described as a "Fructose-
bisphosphate aldolase class 1, from Mezorhizobium sp. (strain BNC1)"

§ It belongs to Pfam PF00274, the Fructose-1,6-Bisphosphate Aldolases

(Class 1); this family contains 24 PDB structures.

§ When we run BlastP on Q11CR8_MESSB against the PDB, we get hits

to all these structures; %ids range from 48 to 54%, which is
reassuringly high.

§ Let’s try SwissModel in Automated mode:

§ http://swissmodel.expasy.org/workspace
 Demonstrations
§ Uniprot entry Q07159 is another Class I FBP Aldolase from
Staphylococcus carnosus. It is unusual, however – most bacterial FBP
Aldolases belong to Class II (PF00596)

§ When we run a Blast against the PDB, again we get hits to all twenty-
four PDB structures, but identities range from 25 to 32% – not good.

§ Try SwissModel in Automated mode...

§ Oh dear, only part of our sequence has been modelled – automated

mode has failed this time. Pfam has a family alignment, so let’s use it
as input to Alignment mode.
 Demonstrations
Now let’s assess our two complete models:
§ Go to [Tools] >> Structure Assessment, and upload each
model in turn:
– Add Whatcheck and Procheck to the selection, and add the
resolution (check result file for automated mode, or check
PDB website for alignment mode)

§ Also, go to the SolvX Server and input our models. It

should give us (at least) two graphs per input – the first will
be our model, the other(s) our template(s).
 Demonstrations
§ What validation checks are the important ones?
• Whatcheck: Accessibility-related (2.6), 3D-Database (2.8),
Geometric (2.5.4 2.5.12 2.5.13 2.5.14) Checks. Make a note
of bad residues and regions.
• Procheck: Residues Report (G-factors) and Ramachandran
Plot. Again, note bad residues and regions.
• SolvX: Compare your model with the template(s). Where are
the worst (most solvent accessible) regions? Note them down.

§ How does your alignment model for ALF1_STACA

compare with your neighbour’s model?