1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Homology Modeling Anne Mølgaard, CBS, BioCentrum, DTU

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Why can we do it? The structure of a protein is uniquely determined by its amino acid sequence (but sequence is sometimes not enough): –prions –pH, ions, cofactors, chaperones Structure is conserved much longer than sequence in evolution

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU How often can we do it? There are currently ~30000 structures in the PDB (but only ~4000 if you include only ones that are not more than 30% identical and have a resolution better than 3.0 Å) An estimate says that ~50% of all sequences have a structurally characterized homolog

4. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Worldwide Structural Genomics ”Fold space coverage” Complete genomes Signaling proteins Improving technology Disease-causing organisms Model organisms Membrane proteins Protein-ligand interactions

5. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Structural Genomics in North America 10 year $600 million project initiated in 2000, funded largely by NIH AIM: structural information on 10000 unique proteins (now 4-6000), so far 1000 have been determined Improve current techniques to reduce time (from months to days) and cost (from $100.000 to $20.000/structure) 9 research centers currently funded (2005), targets are from model and disease-causing organisms (a separate project on TB proteins)

6. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Homology modeling for structural genomics Roberto Sánchez et al. Nature Structural Biology 7, 986 - 990 (2000)

7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU How well can we do it?

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Sali, A. & Kuriyan, J. Trends Biochem. Sci. 22, M20–M24 (1999)

9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU How can we do it? Identify template(s) – initial alignment Improve alignment Backbone generation Loop modeling Side chains Refinement Validation

10. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Template identification Search with sequence –Blast –Psi-Blast –Fold recognition methods Use biological information Functional annotation in databases Active site/motifs

11. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Template quality Selecting the best template is crucial! The best template may not be the one with the highest % id (best p-value…) –Template 1: 93% id, 3.5 Å resolution  –Template 2: 90% id, 1.5 Å resolution

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU 4.0 Å 3.0 Å 1.8 Å 1.0 Å high low Molecules secondary structure elements residues atoms

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Template quality – Ramachandran plot

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Improving the alignment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PHE ASP ILE CYS ARG LEU PRO GLY SER ALA GLU ALA VAL CYS PHE ASN VAL CYS ARG THR PRO --- --- --- GLU ALA ILE CYS PHE ASN VAL CYS ARG --- --- --- THR PRO GLU ALA ILE CYS From ”Professional Gambling” by Gert Vriend http://www.cmbi.kun.nl/gv/articles/text/gambling.html

15. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Backbone generation Generate the backbone coordinates from the template for the aligned regions Several programs can do this, most of the groups at CASP6 use Modeller: http://salilab.org/modeller/modeller.html

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Loop modeling Knowledge based: searches PDB for fragments that match the sequence to be modeled (Levitt, Holm, Baker etc.) Energy based: uses an energy function to evaluate the quality of the loop and minimizes this function by Monte Carlo or MD techniques Combination

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Loops – the Rosetta method Find fragments (10 per amino acid) with the same sequence and secondary structure profile as the query sequence Combine them using a Monte Carlo scheme to build the loop Baker et al

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Side chains Side chain rotamers are dependent on backbone conformation Most successful method in CASP6 was SCWRL by Dunbrack et al: uses a graph-theory knowledge based method to solve the combinatorial problem of side chain modeling http://dunbrack.fccc.edu/SCWRL3.php

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Side chains Prediction accuracy is high for buried residues, but much lower for surface residues –Experimental reasons: side chains at the surface are more flexible –Theoretical reasons: much easier to handle hydrophobic packing in the core than the electrostatic interactions, including H-bonds to waters

20. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Side chains If the seq. id is high, the networks of side chain contacts may be conserved, and keeping the side chain rotamers from the template may be better than predicting new ones

21. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Refinement Energy minimization Molecular dynamics –Big errors like atom clashes can be removed, but force fields are not perfect and small errors will also be introduced – keep minimization to a minimum or matters will only get worse

22. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Error recovery If errors are introduced in the model, they can normally not be recovered at a later step –The alignment can not make up for a bad choice of template –Loop modeling can not make up for a poor alignment If errors are discovered the step when they were introduced should be redone

23. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Validation Most programs will get the bond lengths and angles right The Ramachandran plot of the model usually looks pretty much like the Ramachandran plot of the template (so select a high quality template) Inside/outside distributions of polar and apolar residues can be useful

24. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Validation – ProQ server ProQ is a neural network based predictor that based on a number of structural features predicts the quality of a protein model ProQ is optimized to find correct models in contrast to other methods which are optimized to find native structures Arne Elofssons group: http://www.sbc.su.se/~bjorn/ProQ/

25. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Structure validation ProCheck http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html WhatIf server http://swift.cmbi.kun.nl/WIWWWI//

26. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Homology modeling servers Eva-CM performs continous and automated analysis of comparative protein structure modeling servers A current list of the best performing servers can be found at: http://cubic.bioc.columbia.edu/eva/doc/intro_cm.html

27. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU CASP6 results

28. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU The top 4 homology modeling groups in CASP6

29. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Alfonso Valencia, CASP6 Homology modeling assessment

30. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Dunbrack, Wang & Jin (2004) CASP6 Fold Recognition Assessment The hardest target i CASP6, 8% id