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Semi-automatic structure solution with HKL-3000

Structural Biology. Semi-automatic structure solution with HKL-3000. Structural Biology. Semi -automatic structure solution with HKL-3000. Structural biology bottlneck. I have a dream:

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Semi-automatic structure solution with HKL-3000

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  1. Structural Biology Semi-automatic structure solution with HKL-3000 Bethesda, March 4th 2009

  2. Structural Biology Semi-automatic structure solution with HKL-3000 Bethesda, March 4th 2009

  3. Structural biology bottlneck I have a dream: To convert high throughput, high output SG center into, not necessary high throughput, but very high output center. Can we use sequence to predict: solubility, crystallization, ….. structure, … function, …. Bethesda, March 4th 2009

  4. HKL/HKL-2000 vs. HKL-3000 • HKL/HKL-2000 h, k, l, F, σ Methods in Enzymology (1997) 275: 307-326.  Times cited: 18018 • HKL-3000 partially build model Acta Cryst. D (2006) 62: 859-66.  Times cited: 77 PDB deposits: >710 Bethesda, March 4th 2009

  5. Integrated system – HKL-3000current status • HKL-2000, DENZO, SCALEPACK • SHELXD, SHELXE • MLPHARE, CYCLMLPHARE • DM, REFMAC • SOLVE, RESOLVE • ARP/WARP • STATBUILD • O, COOT,CCP4 Interactive development: Integrated approach shows how the choice in one step influences subsequent steps. Bethesda, March 4th 2009

  6. HKL-3000 six mouse click programEngage your brain ! Bethesda, March 4th 2009

  7. HKL or Automatic structure solution Bethesda, March 4th 2009

  8. Why to engage my brain ?One little detail … O-ring Bethesda, March 4th 2009

  9. IntegrationHKL-3000 is a pipeline component Other systems provide additional contextual up- and downstream information Pepdb expression & purification Xtaldb crystallization HKL-3000 Data collection, diffraction & structure solution Wetlab chemicals & solutions Bethesda, March 4th 2009

  10. Project setup Check against PDB during data collection Bethesda, March 4th 2009

  11. Completeness – overlapsMulti-crystal strategy Bethesda, March 4th 2009

  12. Priceless crystals Crystallography lab Star of Africa Bethesda, March 4th 2009

  13. Scaling Signal from Sulfur Bethesda, March 4th 2009

  14. Feedback of possible hardware, sample or experiment problems • Detector • Goniostat • Cooling • Crystal decay • Experiment • Twinning • ….. Bethesda, March 4th 2009

  15. Possible Pseudo-translational Symmetry Bethesda, March 4th 2009

  16. SAD or MAD phasing Bethesda, March 4th 2009

  17. Substructure solution • Data management in HKL-3000 - substructure solution Bethesda, March 4th 2009

  18. Substructure enantiomorph determination Bethesda, March 4th 2009

  19. Double conformations Bethesda, March 4th 2009

  20. Optimization of solvent content worthwhile but slow manual automatic optimization Bethesda, March 4th 2009

  21. NCS identification and use From sites From map Bethesda, March 4th 2009

  22. Model building and preliminary refinement (10 minutes, 190aa) Unidentified density -> Xtaldb Secondary structure after each cycle Bethesda, March 4th 2009

  23. TM1086 (APC4579) 10 Se-Met/282 aa 35.1kDa I222 a= 290Å, b= 300Å, c=316Å ~81% solvent content = 17 molecules in AU ~38% solvent content = 57 molecules in AU Bethesda, March 4th 2009

  24. TM1086 (APC4579) 282 aa 35.1kDa P3221 a= 165Å, b= 165Å, c=98Å ~78% solvent content = 3 molecules in AU ~40% solvent content = 8 molecules in AU Bethesda, March 4th 2009

  25. TM1086 (APC4579) Bethesda, March 4th 2009

  26. I222 crystal form 30 chains in AU 8460 aa 1.05MDa ~210 Å Bethesda, March 4th 2009

  27. MCSG sequence from electron density map APC81521 ( ChangSoo) Final model with right target APC81521 Attempt to use automatic building with wrong target APC81501 To figure out the right target at the resolution of 2.5Ǻ 1. Manually build helixes and stands; 2. Submit this model to dali to find out several similar structures in PDB; 3. Search targetDB(MCSG) using the sequence of these structures to find out the right target. Bethesda, March 4th 2009

  28. 2as8 1.95Å resolution, R=18.8/17.4, Rfree=25.2/23.1 Bethesda, March 4th 2009

  29. # of PDB deposits with given # of Mg ions # of deposits # of Mg Bethesda, March 4th 2009

  30. Four deposits - 1896 Mg ions Bethesda, March 4th 2009

  31. Unknown ligands Bethesda, March 4th 2009

  32. Completeness of data and completeness of the model (3bqs) Bethesda, March 4th 2009

  33. 3bqs 1ze0 Acta Cryst. D 63(2007)348-54 Bethesda, March 4th 2009

  34. Molecular Replacement SGC Zoledronic acid in Cryptosporidium parvum Arun Malhotra RNase T Bethesda, March 4th 2009

  35. Unique content in ASU [%] = = (1 – solvent content)/NCS factor 70 No methods to interpret such maps Arp/wARP builds most of the model Challenging projects 60 Atomicity based phasing e.g. ME 50 40 30 20 10 2.0 3.5 4.0 3.0 1.5 2.5 Resolution [Å] Directions for a future Using NCS in structure solution Using high-resolution native data with low-resolution phasing data The boundaries can be moved by improving diffraction data quality or by using more elaborate methods Bethesda, March 4th 2009

  36. People involved • Andrzej Joachimiak • Rongguang Zhan • Youngchang Kim • Marianne Cuff • Chris Lazarski and SBC/MCSG staff • Zbyszek Dauter • Tom Terwilliger Solve/Resolve • George Sheldrick Shelxd/Shelxe • Paul Emsley Coot • Garib Murshudov Refmac • Grants: • NIH GM53163, GM62414, GM74942 • NIAID HHSN272200700058C • DOE, NCI, HKL Research Wladek Minor • Marcin Cymborowski • Maks Chruszcz • Matt Zimmerman • Heping Zheng • Marek Grabowski Zbyszek Otwinowski • Dominika Borek • Andrzej Kudlicki Bethesda, March 4th 2009

  37. Possible pseudo-translational symmetry Bethesda, March 4th 2009

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