homology modelling ? x-ray ? nmr ?. homology modelling !

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