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• Well regarded program for Homology/Comparative Modelling.• Current Version 9v2. http://www.salilab.org/modeller/• Requires an Input file, Sequence alignment & Template
structure.
ATOM 1 N ASP A 443 -15.943 41.425 44.702 1.00 44.68 ATOM 2 CA ASP A 443 -15.424 42.618 45.447 1.00 43.15 ATOM 3 C ASP A 443 -14.310 43.306 44.686 1.00 41.81 ATOM 4 O ASP A 443 -14.298 44.528 44.539 1.00 42.61
Template Structure Initial Model (*.ini) Output Model(s) (*.B999*)
Valine Glutamine Change inRotamer
Modeller : Output
• .log : log output from the run.
• .B* : model generated in the PDB format.
• .D* : progress of optimisation.
• .V* : violation profile.
• .ini : initial model that is generated.
• .rsr : restraints in user format.
• .sch : schedule file for the optimisation process.
An Iterative Process
Modeller Features & Restraints• Secondary Structure.
Regions of the protein may be forced to be α-helical or β-strand.
• Distance restraints.The distance between atoms may be restrained.
• Symmetry.Protein multimers can be restrained so that all monomers are identical.
• Disulphide Bridges.Two cysteine residues in the model can be forced to make a cystine bond.
• Ligands.Ions, waters and small molecules may be included from the template.
• Loop Refinement.Regions without secondary structure often require further refinement.
Structural Convergence
• The catalytic triad of Serine, Aspartate and Histidine is found in certain protease enzymes. (a) Subtilisin (b) Chymotrypsin.
• However, the overall structure of the enzyme is often different.
• This is also important when considering ligand binding sites.
Modelling Ligand Interactions• Small molecules, waters and ions
can be retained from the template structure.
• It is possible to search for homologues based on the ligands they bind.
• Experimental data, especially mutagenesis is very useful when modelling ligand binding sites.
• Although the key residues may often remain, the overall structure of the protein may vary radically.
• The presence of the ligand is also likely to alter the conformation of the protein.
1ATN
1E4G
ATP Binding Site
Conformational States
• The backbone structure of the model will be almost identical to that of the template.
• Therefore the conformational state of the template will be retained in the resultant homology model.
• This is important when considering the open or closed conformation of a channel…
• … or the Apo versus bound state of a ligand binding site.
Closed
Open
Loop Modelling
Issues with Loop Modelling
• As loops are less restrained by hydrogen bonding networks they often have increased flexibility and therefore are less well defined.
• In addition the increased mobility make looped regions more difficult to structurally resolve.
• Proteins are often poorly conserved in loop regions.
• There are usually residue insertions or deletions within loops.
• Proline and Glycine resides are often found in loops – we’ll come back to this when discussing Model evaluation protocols.
Loop Modelling
• There are two main methods for modelling loops:1. Knowledge based: A PDB search for fragments that match the sequence to be modelled.
2. Ab initio: A first principles approach to predict the fold of the loop, followed by minimisation steps.
• Many of the newer loop prediction methods use a combination of the two methods.
• These approaches are being developed into methods for computationally predicting the tertiary structure of proteins. eg Rosetta.
• But this is computationally expensive.
• Modeller creates an energy function to evaluate the loop’s quality.
• The function is then minimised by Monte Carlo (sampling), Conjugate Gradients (CG) or molecular dynamics (MD) techniques.
Predicting Sidechain Conformations
• Networks of side chain contacts are important for retaining protein structure.
• Sidechains may adopt a variety of different conformations, but this is dependent on the residue type.
• For example a threonine generally adopts 3 conformations, whilst a lysine may adopt up to 81.
• This is dependent backbone conformation of the residue.
• The different residue conformations are known as rotamers.
• Where a residue is conserved it is best to keep the side chain rotamer from the template than predict a new one.
• Rotamer prediction accuracy is high for buried residues, but much lower for surface residues:– Side chains at the surface are more flexible.– Hydrophobic packing in the core is easier to handle than the electrostatic
interactions with water molecules. (cytoplasmic proteins)
• Most successful method is SCWRL by Dunbrack et al.: http://dunbrack.fccc.edu/SCWRL3.php
Model Evaluation
Initial Options
1. For every model, Modeller creates an objective function energy term, which is reported in the second line of the model PDB file (.B*).
• This is not an absolute measure but can be used to rank models calculated from the same alignment. The lower the value the better.
2. A Cα-RMSD (Root Mean Standard Deviation) between the template structure and models can also be used to compare the final model to its template.
• A good Cα-RMSD will be less than 2Å.
Model EvaluationMore Advanced Options
• Procheck, PROVE, WhatIf:Stereochemical checks on bond lengths, angles and atomic contacts.
• Ramachandran Plot is a major component of the evaluation.
• Ensures that the backbone conformation of the model is normal.
• Modeller is good on the whole, but sometimes struggles with residues found in loops.
• Homology Modelling is a valuable tool for structural biologists.Homology Modelling is a valuable tool for structural biologists.
• There are five main stages:There are five main stages:1.1. Identify an appropriate template structure(s).Identify an appropriate template structure(s).2.2. Create a Sequence alignment.Create a Sequence alignment.3.3. Perform the homology modelling.Perform the homology modelling.4.4. Analyse and Evaluate the quality of the model.Analyse and Evaluate the quality of the model.5.5. Refinement.Refinement.
• It is important to take time when constructing a model – It is important to take time when constructing a model – Crystallography is difficult & time consuming!Crystallography is difficult & time consuming!
• A model should not be rushed and should be fully checked!A model should not be rushed and should be fully checked!
Practical Session• The notes and files for the Practical session can be found at: The notes and files for the Practical session can be found at: http://weblearn.ox.ac.uk/site/medsci/bioch/postgrad/compbio/2007dec/ps/http://weblearn.ox.ac.uk/site/medsci/bioch/postgrad/compbio/2007dec/ps/
• The file name is dtc_homology.tarThe file name is dtc_homology.tar
• Untar the file in your home directory using:Untar the file in your home directory using:$ tar cvf dtc_homology.tartar cvf dtc_homology.tar
• This will produce a folder called DTC, which contains three Exercises.This will produce a folder called DTC, which contains three Exercises.
• There are also two word documents: There are also two word documents: • Homology_Modelling_Practical_07.docHomology_Modelling_Practical_07.doc – Details of the – Details of the
practical.practical.• Homology_Practical_Notes.docHomology_Practical_Notes.doc – For your results. – For your results.
• If you need any help please let me know.If you need any help please let me know.
Practical Session• Details of the Three Exercises:Details of the Three Exercises:
2.2. (a) Retrieve the Sequence of interest.(a) Retrieve the Sequence of interest.(b) Find a Suitable Template.(b) Find a Suitable Template.(c) Modeller Sequence Alignment Generation.(c) Modeller Sequence Alignment Generation.(d) Homology Modelling a Dimer.(d) Homology Modelling a Dimer.
3.3. (a) Homology Modelling a Tetramer with Ligands.(a) Homology Modelling a Tetramer with Ligands.(b) Structural Alignment of Template to Model. (b) Structural Alignment of Template to Model. (c) Visualising Ligand Binding Sites.(c) Visualising Ligand Binding Sites.(d) Computational Mutagenesis.(d) Computational Mutagenesis.