Molecular Replacement: “Practical” Application

1. Richard L. Walter CSO, Shamrock Structures rwalter@shamrockstructures.com Molecular Replacement:“Practical” Application

2. Outline & Objectives • Review the basics of MR: a technique for good or evil? • The truth of applying MR: the theoretical, the real, the ugly • Some “practical” things to try....and why you should have Mass Spectroscopist. Molecular Biologist, and Molecular Modeller friends!

3. } A Crystals OR...Imagining Proteins to be Peanuts/Neck Pillows The Concept of MR A Search Model A correctly rotated Search Model A correctly translated Search Model P …”P” is for PROTEIN

4. A very clear 3 atom protein Patterson... atomic vectors added for clarity Add Some “Patterson Vectors” The Basics of MR: The RotationA “Theoretical View” using “Traditional Methods” A nice, “typical” 3 atom protein structure A Patterson map...looks familiar, but not quite right A nice search model Let's try rotating it We Got it! OK...sure, you have to tumble it in a third rotation (not shown)‏ ...but that's easy...so THIS is EASY!

5. t The Basics of MR: The Translation Correctly rotated molecule sitting at unit cell origin t = 3D translation So, that looks even EASIER!

6. So, MR is EASY...a technique for GOOD! What could POSSIBLY go wrong!

7. One Slide to TOTAL Confusion Two molecules in the cell from a dimer or just crystal symmetry)‏ Let's try a 5 atom protein: Whoa!... That's much more complex ...and it's only 10 atoms! ...a little more confusing, but still OK...I think? What if I don't have ALL the atoms right? What if the rotation is wrong? So, I might get the vector positions correct...but not their magnitudes??? Some vectors STILL overlap!

8. Proteins are Complex • Average residue contains 8 “heavy” atoms • Average protein contains 300 amino acids • Average structure contains 2400 atoms

9. Let's Get back to “PRACTICAL” A Protein An Asymmetric Unit A Unit Cell A Crystal! A “Model” for our Protein Our Hero

10. Revisiting Rotation A Protein Our “body-foot domain” looks good...but something's not quite right about the “head domain” 22º CW Our “head domain” looks good...but now look at the “body-foot domain” 47º CW • So, our model that looked so good may not be so good

11. Revisiting Our Model A Protein Now both our “body-foot” and “head” domains look good...even got some ears! 22º CW An Improved Model • Excellent!...but how would we know to build such a model a priori? A

12. ...And “One” Other Thing You have to find ALL the contents of the AU Our rotation & translation look good...but the cell looks too empty OK...we found a 2nd rotation & translation” …sort of ??? but there's still something wrong? ...AU contents don't have to be “identical” ...AND…they have to pack reasonably!

13. ...But wait, that's not all! ≠ ≠ ≠ because... ≠ ≠ because... There are LOTS of atoms in secondary structural elements which means there are a LOT of resulting Patterson vectors ...RIGHT or WRONG!

14. What We REALLY Learned Happy Bunnies are Insidious & Evil MR is Evil! Why would ANYONE ever do this horrible technique?

15. Now that we have talked about why MR should not work… Perhaps we can talk about how to make it work Because….MR actually DOES work!

16. The Simple Answer to “Practical” SOLUTIONRC 1 21.96 55.01 328.44 0.0000 0.0000 0.0000 14.0 55.6 25.3 19.6 1 SOLUTIONRC 1 9.00 54.87 327.31 0.0000 0.0000 0.0000 8.0 57.1 14.2 11.0 2 SOLUTIONRC 1 39.10 75.66 28.54 0.0000 0.0000 0.0000 7.4 57.5 13.6 10.8 3 SOLUTIONRC 1 21.50 28.68 43.50 0.0000 0.0000 0.0000 7.5 57.3 15.1 10.0 4 SOLUTIONRC 1 61.63 76.42 43.19 0.0000 0.0000 0.0000 8.2 56.8 14.4 9.9 5 SOLUTIONRC 1 71.12 48.16 211.00 0.0000 0.0000 0.0000 8.4 57.0 14.4 9.8 6 SOLUTIONRC 1 59.98 50.91 330.51 0.0000 0.0000 0.0000 8.0 57.2 15.1 9.8 7 • If you see this…You are golden • If you do NOT see this….GIVE UP! • Just kidding…sort of!

17. Why You Often Can “See This” Resolution Fold Conservation Example 1 Example 2 ALL TYROSINE KINASES DOMAINS!

18. Why Resolution Helps X-ray Data between 3.5 – 6Å will do Anything higher slows you down or even hurts you …than to match these Much easier to match these

19. 18% Identity 15% Identity It’s all about the starting Structure The 3D Structure …NOT the Amino Acid Sequence So how do you get a good starting structure???

20. Option 1: Try a simple BLAST • All solved structures (sure!) are deposited in the Protein Data Bank (http://www.rcsb.org/pdb/) • BLAST your amino acid sequence (or a Swiss-Prot accession number) against the PDB structure database: • Try http://expasy.org/tools/blast/ or http://www.ncbi.nlm.nih.gov/BLAST/ • 20%+ sequence identity usually means similar 3-D structures.

21. Option 2: Structural Overlap • Take a diverse subset of your BLAST results. • Structurally overlap this subset using any number of available tools: • Most graphics programs: Quanta, Coot, etc. • On-line servers for 3D structure comparison: Combinatorial Extension (http://cl.sdsc.edu/), Dali (http://www.ebi.ac.uk/dali/), a good comprehensive list is at (http://en.wikipedia.org/wiki/Structural_alignment_software). • Look for a highly conserved core and try several of the structures that closely match it or trim some of the structures down to this core.

22. Option 3: Model Guided Structure ID • Submit your sequence to a threader (e.g., 3D Jigsaw:http://www.bmm.icnet.uk/servers/3djigsaw/ ; FUGUE:http://tardis.nibio.go.jp/fugue/prfsearch.html)or similar model building server. • Many databases and servers of programs exist: (http://mbcf.dfci.harvard.edu/cmsmbr/biotools/biotools9.html\) (http://www2.uah.es/biomodel/pe/protexpl/psbiores.htm ) • My personal favorite is the Meta server athttp://bioinfo.pl/ • Throw the models themselves away but pay attention to what PDB files were used to construct the models. • Make a list of the top 20 – 30 PDB files that were used most frequently and structurally overlap them • Repeat “Option 2” with this test set

23. Option 4: Make Friends with MS • Run your protein on an SDS-PAGE gel. • Give the gel to a skilled Mass Spectroscopist and have her/him cut out the band, tryptic digest the extracted band, and run LS-MS. • Have your MS friend run the tryptic fragment map against his/her database of such digests. • Take the list of proteins IDed by the MS mapping and BLAST them against the PDB, repeating Option 1, 2, and 3 as necessary with these “hits”. • This is a great way to quickly get the structure of a protein if you don’t even know the sequence!

24. Option Last: Build Homology Models • Take the models that were generated by “Option 2” out of the garbage and use them in MR attempts. • Build homology models by any other standard method that you or (preferrably) a skilled modeler friend of yours uses. • Set up heavy atoms soaks; see if you have enough sulfur anomalous single; hope that yours is an unrecognized metallo-enzyme with a reachable edge; undertake MAD.

25. A Final Caveat P41212 SOLUTIONTF1 1 21.96 55.01 328.44 0.0073 0.2403 0.2567 41.7 45.2 41.4 1 27.7 SOLUTIONTF1 1 9.00 54.87 327.31 0.8385 0.2499 0.1907 18.9 54.9 20.3 3 10.9 SOLUTIONTF1 1 39.10 75.66 28.54 0.8766 0.7239 0.1881 18.0 55.2 19.7 10 22.3 SOLUTIONTF1 1 21.50 28.68 43.50 0.8838 0.4984 0.2995 18.1 55.4 20.7 2 16.1 SOLUTIONTF1 1 61.63 76.42 43.19 0.9739 0.2338 0.3070 19.1 54.3 20.1 6 23.7 SOLUTIONTF1 1 71.12 48.16 211.00 0.7945 0.2326 0.1719 19.3 54.8 20.5 4 6.7 SOLUTIONTF1 1 59.98 50.91 330.51 0.6805 0.6670 0.0686 17.5 55.1 20.6 4 5.4 Great Solution!! P43212 SOLUTIONTF1 1 21.96 55.01 328.44 0.0063 0.7394 0.0000 69.1 34.9 70.5 1 28.5 SOLUTIONTF1 1 9.00 54.87 327.31 0.5901 0.4253 0.4508 19.4 55.5 19.2 6 15.4 SOLUTIONTF1 1 39.10 75.66 28.54 0.1019 0.3282 0.2175 18.0 55.3 21.0 2 24.3 SOLUTIONTF1 1 21.50 28.68 43.50 0.7073 0.4348 0.4263 17.7 55.2 21.1 3 20.8 SOLUTIONTF1 1 61.63 76.42 43.19 0.2749 0.6853 0.3370 17.9 55.0 18.9 1 8.0 SOLUTIONTF1 1 71.12 48.16 211.00 0.9809 0.4550 0.0000 18.2 55.4 18.9 2 11.0 SOLUTIONTF1 1 59.98 50.91 330.51 0.3996 0.5807 0.1555 17.5 55.6 21.3 1 7.5 Even Better Solution?? Don’t throw away what seems like a guaranteed MR solution because the maps look like crap: make sure that you checked ALL enantiomorphic space groups!

26. Help “In Theory” & “In Image”: A Thanks • Artem Evdokimov – Pfizer, Inc. • Bobby Barnett – U Cincinnati • David Wishart – U Alberta • Steve Hubbard – Skirball Institute • Bart Hazes – U Alberta • Randy Read – U Cambridge • Michael Rossmann – Purdue U