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Homology modelling ?Homology modelling ?
X-ray ?
NMR ?
Homology Modelling !Homology Modelling !
Helices are HelicesHelices are Helices
Strands are StrandsStrands are Strands
Structure=ConservationStructure=Conservation
Structure=ConservationStructure=Conservation
Modelling beats X-rayModelling beats X-ray
Data ~ infinite…Data ~ infinite…
What can be Modelled ?What can be Modelled ?
What can be Modelled ?What can be Modelled ?
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
Template detectionTemplate detection
Normally BLAST is good enough.If BLAST doesn’t find a template,You should not want to build a model.When desperate, use PSI-BLAST (on PDB + SwissProt), or use threading.
ThreadingThreading
Threading means:Use information from the template structure to detect homology, or to improve an alignment.
ThreadingThreading
Small residues
ThreadingThreading
Alcoholic residues
ThreadingThreading
The folded ‘protein’
ThreadingThreading
Two aligned ‘proteins’
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
AlignmentAlignment
Run BLAST on model sequenceRun BLAST on template sequenceSelect 50-100 representativesDo multiple sequence alignmentKeep only model and template
How to align:How to align:
ASASASASASAS
YPYPYPYPYPYP
(three ways…)
How to align:How to align:
ASASASASASAS-AYAYAYAYAYAY--YPYPYPYPYPYP
(two ways…)
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
Alignment optimizationAlignment optimization
1 Use threading techniques.2 Shift gaps around:
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
Select ‘best’ templateSelect ‘best’ template
Deal with errorsDeal with errors
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
Exchange side chainsExchange side chains
Keep template rigidDetermine best rotamerDo NOT optimize rotamersIf best rotamer doesn’t fit, start thinking.If the model is bad, you had the wrong template, or the wrong alignment.Make sure your model exists…
Position specific rotamersPosition specific rotamers
Position specific rotamersPosition specific rotamers
Position specific rotamersPosition specific rotamers
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
Insertions - DeletionsInsertions - Deletions
Insertions are impossible
Deletions:Move gap around in template till end point distance is short. If this is not possible, you have either the wrong template, or the wrong alignment.
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
Model OptimizationModel Optimization
Do NOT use molecular dynamics:
Model OptimizationModel Optimization
Use 25 – 50 steps energy minimization, or use a force field that has been especially designed for the optimization of homology models.
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate
The ‘8’ Steps of ModellingThe ‘8’ Steps of Modelling
1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate