©CMBI 2002 Homology modelling ? X-ray ? NMR ? Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate.

©CMBI 2002

Homology modelling ?

X-ray ?

NMR ?IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate

©CMBI 2002

Homology Modelling !

IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate

©CMBI 2002

Helices are Helices


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Strands are Strands


©CMBI 2002

Structure=Conservation


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Structure=Conservation


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Modelling beats X-ray


©CMBI 2002

Data ~ infinite…


Copied from TIBS special volume on bioinformatics

©CMBI 2002

What can be Modelled ?


©CMBI 2002

What can be Modelled ?


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The ‘8’ Steps of Modelling

1 Detect template2 Get alignment3 Optimize alignment4 Optimize template5 Exchange side chains6 Deal with insertions/deletions7 Optimize model8 Validate9 Iterate


©CMBI 2002

Template detection

Normally BLAST is good enough.

If BLAST doesn’t find a template, you should not want to build a model anyway.

When desperate, use PSI-BLAST (on PDB + SwissProt), or use threading.


©CMBI 2002

Threading

Threading means using information from the template structure to:

•detect homology•to improve an alignment.


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Threading

Small residues


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Threading

Alcoholic residues


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Threading

The folded ‘protein’


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Threading

Two aligned ‘proteins’


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Alignment

BLAST the model sequence.BLAST the template sequence.Select 50-100 representatives.Do multiple sequence alignment.Keep only model and template from that multiple sequence alignment.


©CMBI 2002

How to align:

ASASASASASAS

YPYPYPYPYPYP

(three ways…)


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How to align:

ASASASASASAS-AYAYAYAYAYAY--YPYPYPYPYPYP

(two ways…)


©CMBI 2002

Alignment optimization

1 Use threading techniques.2 Shift gaps around (indels):


©CMBI 2002

Select ‘best’ template


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Deal with errors


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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 can exist.


©CMBI 2002

Position specific rotamers


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Insertions - Deletions

Insertions are impossibleDeletions: 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.


©CMBI 2002

Model Optimization

Do NOT use molecular dynamics (unless you know that you know what you are doing):


©CMBI 2002

Model Optimization

Use 25 – 50 steps energy minimization, or use a force field that has been especially designed for the optimization of homology models.


©CMBI 2002

Validation

Use the WHAT_CHECK server. (Next seminar).Use your brains.Compare with structures.Compare with Meta-server.


©CMBI 2002

The ‘8’ Steps of Modelling

Depending on the WHAT_CHECK result, you can restart at each of the previous steps in the modelling process.


©CMBI 2002 Homology modelling ? X-ray ? NMR ? Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate.

Documents

threading threading

threading alignment

steps of modelling

chain indels

template structure

homology modelling

template detection

bioinformatics slide