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

2. ©CMBI 2002 Homology Modelling ! Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

3. ©CMBI 2002 Helices are Helices Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

4. ©CMBI 2002 Strands are Strands Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

5. ©CMBI 2002 Structure=Conservation Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

6. ©CMBI 2002 Structure=Conservation Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

7. ©CMBI 2002 Modelling beats X-ray Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

8. ©CMBI 2002 Data ~ infinite… Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate Copied from TIBS special volume on bioinformatics

9. ©CMBI 2002 What can be Modelled ? Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

10. ©CMBI 2002 What can be Modelled ? Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

11. ©CMBI 2002 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 Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

12. ©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. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

13. ©CMBI 2002 Threading Threading means using information from the template structure to: detect homology to improve an alignment. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

14. ©CMBI 2002 Threading Small residues Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

15. ©CMBI 2002 Threading Alcoholic residues Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

16. ©CMBI 2002 Threading The folded ‘protein’ Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

17. ©CMBI 2002 Threading Two aligned ‘proteins’ Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

18. ©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. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

19. ©CMBI 2002 How to align: ASASASASASAS YPYPYPYPYPYP (three ways…) Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

20. ©CMBI 2002 How to align: ASASASASASAS- AYAYAYAYAYAY- -YPYPYPYPYPYP (two ways…) Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

21. ©CMBI 2002 Alignment optimization 1 Use threading techniques. 2 Shift gaps around (indels): Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

22. ©CMBI 2002 Select ‘best’ template Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

23. ©CMBI 2002 Deal with errors Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

24. ©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. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

25. ©CMBI 2002 Position specific rotamers Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

26. ©CMBI 2002 Position specific rotamers Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

27. ©CMBI 2002 Position specific rotamers Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

28. ©CMBI 2002 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. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

29. ©CMBI 2002 Model Optimization Do NOT use molecular dynamics (unless you know that you know what you are doing): Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

30. ©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. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

31. ©CMBI 2002 Validation Use the WHAT_CHECK server. (Next seminar). Use your brains. Compare with structures. Compare with Meta-server. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate

32. ©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. Intro Proteins Modelling 8 Steps Detect Threading Alignment Template Side chain Indels Optimize Validate Iterate