©cmbi 2002 homology modelling ? x-ray ? nmr ? intro proteins modelling 8 steps detect threading...
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©CMBI 2002
Homology modelling ?
X-ray ?
NMR ?IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Homology Modelling !
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Helices are Helices
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Strands are Strands
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Structure=Conservation
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Structure=Conservation
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Modelling beats X-ray
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Data ~ infinite…
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
Copied from TIBS special volume on bioinformatics
©CMBI 2002
What can be Modelled ?
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
What can be Modelled ?
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
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
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©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.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Threading
Threading means using information from the template structure to:
•detect homology•to improve an alignment.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Threading
Small residues
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Threading
Alcoholic residues
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Threading
The folded ‘protein’
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Threading
Two aligned ‘proteins’
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
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.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
How to align:
ASASASASASAS
YPYPYPYPYPYP
(three ways…)
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
How to align:
ASASASASASAS-AYAYAYAYAYAY--YPYPYPYPYPYP
(two ways…)
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Alignment optimization
1 Use threading techniques.2 Shift gaps around (indels):
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Select ‘best’ template
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Deal with errors
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
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.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Position specific rotamers
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Position specific rotamers
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Position specific rotamers
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
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.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Model Optimization
Do NOT use molecular dynamics (unless you know that you know what you are doing):
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©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.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©CMBI 2002
Validation
Use the WHAT_CHECK server. (Next seminar).Use your brains.Compare with structures.Compare with Meta-server.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate
©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.
IntroProteinsModelling8 StepsDetectThreadingAlignmentTemplateSide chainIndelsOptimizeValidateIterate