1 / 43

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

sal
Download Presentation

Two parts to successful model building

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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 f and y angles. PATTERN RECOGNITION SKILLS Recognizing structural features in electron density maps and skeleton maps. .a-helix density. .b-strand density. Recognize a-carbons positions Recognize side chain density Two parts to successful model building

  2. an amino acid residue Ni+1 C O Ca Og Cb N Oi-1 carbon nitrogen oxygen Basic protein chemistry: Phi(f) and Psi(Y) planar peptides chiral a-carbons allowed f,y angles- seeRamachandran plot

  3. Lowest energy f,y angles correspond to a-helices and b-sheets b-sheet a-helix Ramachandran plot Lets focus on recognizing helix and strand features in electron density maps.

  4. 90o Be able to recognize a helix from different perspectives Viewed down helical axis Viewed perpendicular to helical axis

  5. The hole through the center of a helix is the most distinctive feature of a-helix density. 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. Viewed perpendicular to helical axis

  6. 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. But, be sure to view both perspectives when modeling an a-helix. When viewed down the helical axis. Both orientations of the helix appear to fit the electron density OK. Correct Incorrect

  7. 10 9 8 7 6 5 4 3 2 1 Helix directionality C-terminus 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? Backbone oxygens Backbone nitrogens N-terminus

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

  9. Cb Cb Cb Cb Cb Cb Cb Cb Cb Cb Cb Cb Cb point to N-terminus Cb Cb Cb Cb Cb Cb Cb Cb Cb point to the N-terminus like the branches of a Christmas tree.

  10. Cb Cb Cb Cb Cb Cb Cb Cb Cb Cb Cb point to the N-terminus like the branches of a Christmas tree.

  11. Which way is the C-terminus pointing?

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

  13. 4.8 Å 4.8 Å 4.8 Å b-strands appear as parallel tubes of density spaced 4.8 Å apart. b-strands are never alone. STRAND1 STRAND 2 STRAND 3 STRAND 4

  14. 1 2 3 4 5 6 7 8 9 10 b-strands also have directionality From this perspective, side chains of successive residues alternate in and out of the plane of this page. N-terminus C-terminus

  15. 2 1 1 2 3 3 4 4 6 6 5 5 8 7 8 7 10 10 9 9 90o b-strands viewed from different perspectives From this perspective, side chains of successive residues alternate in and out of the plane of this page. N-terminus C-terminus N-terminus C-terminus 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.

  16. But, be sure to view both perspectives when modeling a b-strand. When viewed using the zig-zag perspective. Both orientations of the strand appear to fit the electron density OK. incorrect correct 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.

  17. 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

  18. Phe - Thr - Ala- Ser or or Val Cys 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.

  19. 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

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

  21. Load Coordinates Open Coordinates Auto Open MTZ Open MTZ, mmcif, fcf, or phs • 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)

  22. siras-pcmbs.phs LoadStructure Factors (map) Open Coordinates Auto Open MTZ Open MTZ, mmcif, fcf, or phs • 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

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

  24. Mutate & Autofit

  25. Select amino acid type

  26. If rotamer is incorrect, choose another

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

  28. Sample different rotamers Accept

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

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

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

  32. What is the next residue?

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

  34. 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

  35. 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.

  36. X X X 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. NO! drag corner

  37. Save coordinates frequently or suffer set backs.

  38. 1 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

  39. sawaya-coot-1.pdb 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

  40. 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.

  41. Results 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 Rwork and Rfree. • 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

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

More Related