2. Two parts to successful model building BUILDING TOOLS –how to use Coot –Initiate trace of protein chain (“Place helix here”) –Test sidechain assignments. Mutate+autofit Select rotamers –Maintain proper geometry “Regularize” bond lengths and angles, planes, eliminate steric clash. “Real space refine” to adjust fit to e- density –Validation tools to detect disallowed  and  angles. PATTERN RECOGNITION SKILLS –Recognizing structural features in electron density maps and skeleton maps..  -helix density..  -strand density. Recognize  -carbons positions Recognize side chain density

3. Basic protein chemistry: Phi(  ) and Psi(  ) planar peptides chiral  -carbons allowed  angles- see Ramachandran plot CC CC OO C O N N i+1 O i-1 carbon nitrogen oxygen an amino acid residue

4. Lowest energy  angles correspond to  -helices and  -sheets  -helix  -sheet Lets focus on recognizing helix and strand features in electron density maps. Ramachandran plot

5. Be able to recognize a helix from different perspectives Viewed down helical axis Viewed perpendicular to helical axis 90 o

6. Viewed perpendicular to helical axis Viewed down helical axis Often it is easier to recognize helical density when viewed down the helical axis due to the distinctive hole through the center of the helix. The hole through the center of a helix is the most distinctive feature of  -helix density.

7. But, be sure to view both perspectives when modeling an  -helix. When viewed down the helical axis. Both orientations of the helix appear to fit the electron density OK. But, when viewed perpendicular to the helical axis, it becomes clear that only one direction of the helix fits the carbonyl bumps in the electron density. Incorrect Correct

8. Helix directionality 1 2 3 4 5 6 7 8 9 10 Protein models are always numbered from N-terminus to C-terminus as shown here. So it is easy to tell the directionality of a helix. But, electron density isn’t labeled with residue numbers. What structural features are present in the electron density map to help you determine which direction to place the helix? C-terminus N-terminus Backbone nitrogens Backbone oxygens

9. Carbonyl oxygens point to C-terminus 1 2 3 4 5 6 7 8 9 10 C-terminus Look for carbonyl bumps

10. C  point to the N-terminus like the branches of a Christmas tree. CC CC CC CC CC CC CC CC CC CC C  point to N-terminus CC CC CC CC CC CC CC CC CC CC

11. C  point to the N-terminus like the branches of a Christmas tree. CC CC CC CC CC CC CC CC CC CC

12. Which way is the C-terminus pointing?

13. Test 2: Which way is the C- terminus pointing ?

14.  -strands appear as parallel tubes of density spaced 4.8 Å apart.  -strands are never alone. STRAND 1 STRAND 2 STRAND 3 STRAND 4 4.8 Å

15.  -strands also have directionality N-terminus C-terminus 13579 246810 From this perspective, side chains of successive residues alternate in and out of the plane of this page.

16.  -strands viewed from different perspectives N-terminus C-terminus 13579 246810 N-terminus C-terminus From this perspective, side chains of successive residues alternate in and out of the plane of this page. 90 o From this perspective, side chains of successive residues alternate up and down. Often it is easier to recognize a beta strand by this distinctive zig-zag pattern than by the pattern shown above. 246810 13579

17. But, be sure to view both perspectives when modeling a  -strand. When viewed using the zig-zag perspective. Both orientations of the strand appear to fit the electron density OK. correct incorrect But, when viewed perpendicular to the zig-zag perspective, it becomes clear that only one direction of the strand fits the carbonyl bumps in the electron density.

18. Proteinase K (Tritirachium album) amino acid sequence 001_MAAQTNAPWG_LARISSTSPG_TSTYYYDESA_GQGSCVYVID 041_TGIEASHPEF_EGRAQMVKTY_YYSSRDGNGH_GTHCAGTVGS 081_RTYGVAKKTQ_LFGVKVLDDN_GSGQYSTIIA_GMDFVASDKN 121_NRNCPKGVVA_SLSLGGGYSS_SVNSAAARLQ_SSGVMVAVAA 161_GNNNADARNY_SPASEPSVCT_VGASDRYDRR_SSFSNYGSVL 201_DIFGPGTSIL_STWIGGSTRS_ISGTSMATPH_VAGLAAYLMT 241_LGKTTAASAC_RYIADTANKG_DLSNIPFGTV_NLLAYNNYQA

19. Assigning the sequence Find a stretch of 5-10 residues with well defined side chain density. Find which amino acid best fits the density by trial and error. (Mutate & Autofit) Keep in mind some residues are isosteric. For example threonine and valine. Check the proteinase K amino acid sequence for a matching sequence of residues. Phe - Thr - Ala- Ser or or Val Cys

20. Proteinase K (Tritirachium album) amino acid sequence 001_MAAQTNAPWG_LARISSTSPG_TSTYYYDESA_GQGSCVYVID 041_TGIEASHPEF_EGRAQMVKTY_YYSSRDGNGH_GTHCAGTVGS 081_RTYGVAKKTQ_LFGVKVLDDN_GSGQYSTIIA_GMDFVASDKN 121_NRNCPKGVVA_SLSLGGGYSS_SVNSAAARLQ_SSGVMVAVAA 161_GNNNADARNY_SPASEPSVCT_VGASDRYDRR_SSFSNYGSVL 201_DIFGPGTSIL_STWIGGSTRS_ISGTSMATPH_VAGLAAYLMT 241_LGKTTAASAC_RYIADTANKG_DLSNIPFGTV_NLLAYNNYQA 001_MAAQTNAPWG_LARISSTSPG_TSTYYYDESA_GQGSCVYVID 041_TGIEASHPEF_EGRAQMVKTY_YYSSRDGNGH_GTHCAGTVGS 081_RTYGVAKKTQ_LFGVKVLDDN_GSGQYSTIIA_GMDFVASDKN 121_NRNCPKGVVA_SLSLGGGYSS_SVNSAAARLQ_SSGVMVAVAA 161_GNNNADARNY_SPASEPSVCT_VGASDRYDRR_SSFSNYGSVL 201_DIFGPGTSIL_STWIGGSTRS_ISGTSMATPH_VAGLAAYLMT 241_LGKTTAASAC_RYIADTANKG_DLSNIPFGTV_NLLAYNNYQA

21. Some amino acids have distinctive shapes, others are isosteric. When in doubt, consider the protein environment.

22. Load Coordinates File -> Open Coordinates--browse window opens –Click “filter” (will show only.pdb files) –Click “sort by date” (will place the lastest coordinates at the top of browser) –Select the.pdb file (e.g. sawaya-coot-0.pdb) Open Coordinates Auto Open MTZ Open MTZ, mmcif, fcf, or phs

23. File -> Open mtz, mmcif, fcf, or phs –Click “filter” (will show only.mtz or.hkl or.phs files) –Click “sort by date” (will place the lastest.phs file at the top of browser) –Select the.phs file siras-pcmbs.phs Open Coordinates Auto Open MTZ Open MTZ, mmcif, fcf, or phs Load Structure Factors (map) siras-pcmbs.phs

24. Zoom in on a distinctive side chain. Calculate ->Model/Fit/Refine

25. Mutate & Autofit

26. Select amino acid type

27. If rotamer is incorrect, choose another

28. CC H H H H H energetically preferred rotation angles about single bonds in side chains CC CC CC N C O Rotamers CC N O=C H H H Nearly eclipsed. Unfavored. CC N O=C H H H A rotamer is energetically favorable because it is one of 3 possible staggered conformations. Newman Projections N O=C H CC H H H H H H N H

29. Sample different rotamers Accept

30. Real Space Refinement can tidy up. If you don’t like it, Reject it. If you accidentally accepted, then, “undo”.

31. If molecule explodes, adjust refinement weight. Lower numbers tighten geometric restraints 5.0

32. Move to next residue. Mutate & Auto Fit.

33. What is the next residue?

34. What is the sequence of these three amino acids? F T A Or F V A ?????

35. Proteinase K (Tritirachium album) amino acid sequence 001_MAAQTNAPWG_LARISSTSPG_TSTYYYDESA_GQGSCVYVID 041_TGIEASHPEF_EGRAQMVKTY_YYSSRDGNGH_GTHCAGTVGS 081_RTYGVAKKTQ_LFGVKVLDDN_GSGQYSTIIA_GMDFVASDKN 121_NRNCPKGVVA_SLSLGGGYSS_SVNSAAARLQ_SSGVMVAVAA 161_GNNNADARNY_SPASEPSVCT_VGASDRYDRR_SSFSNYGSVL 201_DIFGPGTSIL_STWIGGSTRS_ISGTSMATPH_VAGLAAYLMT 241_LGKTTAASAC_RYIADTANKG_DLSNIPFGTV_NLLAYNNYQA

36. Extend N and C termini one amino acid at a time Center on the N or C- terminus of the helix Click on “ Add Terminal Residue” Accept or drag to better location.

37. Remember Do not use maximize button to expand the Coot window to full screen mode. It will hide “pop up” dialog boxes. –Coot will be waiting for a response, but you’ll never see the question because it is hidden behind the full screen window. –Instead, stretch window by dragging corner. X NO! X drag corner X

38. Save coordinates frequently or suffer set backs.

39. Saving first set of coordinates. File menu –Save coordinates Select which coordinate set you want to save. –1 Helix Auto suggestion: –Helix-coot-0.pdb Change to: –sawaya-coot-0.pdb version number 1

40. Updated coordinates? Save with incremented version number. Select save. –1 sawaya-coot-0.pdb Auto suggestion: sawaya-coot-1.pdb Accept. Next time: –sawaya-coot-2.pdb, –sawaya-coot-3.pdb, etc. 1 sawaya-coot-0.pdb sawaya-coot-1.pdb

41. Save coordinates frequently 1) Every 5 minutes. 2) When you have done some modeling that you are especially pleased with. 3) When you are fear that the next step is going to destroy your previous work.

42. Results sawaya1-coot-0.pdb sawaya1-coot-1.pdb sawaya1-coot-2.pdb sawaya1-coot-3.pdb sawaya1-coot-4.pdb sawaya1-coot-5.pdb sawaya1-coot-6.pdb sawaya1-coot-7.pdb sawaya1-coot-8.pdb The last version number of each baton build session represent your best effort at modeling that segment of the protein chain. Next week, we will concatenate these segments of chain into one file and refine them, calculate R work and R free.

43. To control objects Rotate around x or y Translate in x or y