Homology modelling ? X-ray ? NMR ?

Homology Modelling !

Helices are Helices

Strands are Strands

Structure=Conservation

Structure=Conservation

Modelling beats X-ray

Data ~ infinite…

What can be Modelled ?

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Template 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.

Threading Threading means: Use information from the template structure to detect homology, or to improve an alignment.

Threading Small residues

Threading Alcoholic residues

Threading The folded ‘protein’

Threading Two aligned ‘proteins’

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Alignment Run BLAST on model sequence Run BLAST on template sequence Select representatives Do multiple sequence alignment Keep only model and template

How to align: ASASASASASAS YPYPYPYPYPYP (three ways…)

How to align: ASASASASASAS- AYAYAYAYAYAY- -YPYPYPYPYPYP (two ways…)

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Alignment optimization 1 Use threading techniques. 2 Shift gaps around:

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Select ‘best’ template

Deal with errors

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Exchange side chains Keep template rigid Determine best rotamer Do NOT optimize rotamers If 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 rotamers

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Insertions - 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 Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

Model Optimization Do NOT use molecular dynamics:

Model 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 Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate

The ‘8’ Steps of Modelling 1 Detect template 2 Get alignment 3 Optimize alignment 4 Optimize template 5 Exchange side chains 6 Deal with insertions/deletions 7 Optimize model 8 Validate 9 Iterate