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Tutorial
Homology Modelling
A Brief Introduction to Homology
Modeling
Sequence-Structure-Function
Relationships
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Proteins of similar sequences fold into similar
structures and perform similar biological
functions.
The protein sequence has the intrinsic
information to encode the protein structure.
The Noble Prize in Chemistry 1972
Christian B Anfinsen
"for his work on
ribonuclease, especially
concerning the connection
between the amino acid
sequence and the
biologically active
conformation"
The protein sequence is sufficient to specify its 3D
structure
From Nobel Lecture, December 11, 1972, by Christian Anfinsen
Sequence->Structure->Function
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Widespread Automated DNA sequencing =>
more sequence data than structure data
Semi-Automated pipeline of structure
determination is still not widespread.
Nevertheless, structure is more conserved than
sequence.
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Sequence homologs => structural homologs
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See Chapter 9, Baxevanis and Ouellette 3rd edn.
Protein Structure Prediction vs Experimental
Determination
From Chapter 9, Bryan Bergeron, Bioinformatics Computing, 2003 Pearson Education, Inc.
Structure Prediction from sequence
1.Homology (or comparative) modelling
2.Threading
3.Ab initio calculations
Homology modelling is most accurate and
powerful
What is Homology Modeling?
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Homology modeling also known as comparative
modeling uses homologous sequences with known
3D structures for the modelling and prediction of
the structure of a target sequence.
Homology modeling is one of the most best
performing prediction methods that gives
“accurate” predicted models.
How is Homology Modeling done
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Multistep process involves many steps such as:
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Sequence alignment of target/query/unknown protein
sequence to homologous sequence with a known
structure
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structure modification of backbone
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side chain replacements
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Energy minimisation for refinement of structural
model
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Validation of model with visual inspection and etc
Why Homology Modeling?
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The number of protein structures solved so far are fewer than
the number of genes known.
Proteins of biological interest with their orthologous proteins
solved by X-ray crystallography or NMR can be modeled.
Homology modeling is an important method used to predict
the structures of membrane proteins, ion channels,
transporters that are large and difficult to crystallize.
Examples: GPCR (G Protein-coupled receptor), cytochrome
P450 etc.
Overview of the process of Homology
Modeling
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A target sequence (the structure to be predicted)
Identify the homologous sequence with known 3D as
template
Using homology modeling software such as Modeller for
structure prediction (from the Sali Lab)
Model evaluation and refinement
Pre-Modeling Stage:
Template Identification
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Target sequence in FASTA format as input
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Blastp against PDB
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Identify proteins with “good” hit
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Pairwise or multiple sequence alignment
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Further editing the alignment results
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Realign and identify the “good” structural template
Pre-Modeling Stage:
Preparing the Input Files for Modeller
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PDB files for structural templates is required
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The PIR file from the alignment results
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The script file model.top to execute the Modeller program
(latest versions use Python scripts)
In the Heart of Modeller
From the Modeller manual
Evaluation of Predicted Model
Garbage in-Garbage out
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The predicted model can be superimposed with
known structure determined by experiment
http://wishart.biology.ualberta.ca/SuperPose/
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The predicted model is normally evaluated by root
mean square deviation (RMSD)
From http://swissmodel.expasy.org//course/text/chapter6.htm
Calculating RMSD
•
N = number of atoms, d = the distance in Angstrom between corresponding atoms in the
experimental and predicted protein structures.
From Chapter 9, Bryan Bergeron, Bioinformatics Computing, 2003 Pearson Education, Inc.
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Some Rule of Thumb for Structural Modelling
Proteins that share 35 to 50% sequence identity with their
templates, will generally deviate by 1.0 to 1.5 Å from their
experimental counter parts.
Crystallographic structures of identical proteins can vary
not only because of experimental errors and differences in
data collection conditions and refinement, but also because
of different crystal lattice contacts and the presence or
absence of ligands.
Quality of Model
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The correctness of a model is essentially determined by the quality
of the sequence alignment used to identify the template.
If the sequence alignment is wrong in some regions, then the
spatial arrangement of the residues in this portion of the model will
be incorrect.
Viewing the Model
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The predicted model is saved in PDB format that can be
viewed by molecular visualizing software such as
Rasmol, PyMol, MolMol, Sybyl etc.
Viewing is an essential step to validate the quality of the
predicted model.
In this practical, Rasmol is used to view the predicted
structure.
Model Refinement
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Gaps in sequence alignment represent insertion/deletion
regions of target. Loop modeling is used to refine these
regions (not cover in this practical)
The predicted model can be further refined by energy
minimization to remove unfavourable non-bonded
contacts with force fields such as CHARMM, AMBER
or GROMOS etc (not covered in this practical)
Web-Based Homology Modeling: The
SWISS-MODEL Server
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The aim of the Internet-based SWISS-MODEL
server is to provide a comparative protein modelling
tool independent from expensive computer hardware
and software.
http://www.expasy.ch/swissmod/SWISS-MODEL.html
Steps involved in SwissModel
http://swissmodel.expasy.org/
1.Take target sequence of unknown structure
2.Using BLAST to select closest homolog with known
structure as structural template
http://swissmodel.expasy.org/SM_Blast.html
3.Insert target sequence and homologous sequence to
Web service
http://swissmodel.expasy.org/SM_FIRST.html
4.Results will be emailed back to you.
5.Warning: Structure needs to be analysed and
validated
Simple Homology Modelling using
Modeller
1. Take target sequence of unknown structure
2. Using BLAST to select closest homolog with known
structure.
3. Using Clustalx or Jalview to do pairwise alignment between
target sequence and structural homolog and manual
adjustment
4. Inspection of missing structural features in structural
homolog
5. Preparation of alignment file align.pir
6. Use Modeller7v7 software (http://salilab.org/modeller/) to
do the homology modelling
Structure Validation
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Visual inspection
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Minimise torsion angles in disallowed regions of
Ramachandran plots
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Maximised hydrogen bonding
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Minimised exposed hydrophobic residues
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Packing etc.
Analysis – e.g. run Procheck
(http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html),
VADAR, Verify3D etc