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

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Presentation on theme: "Bethesda, March 4 th 2009 Semi-automatic structure solution with HKL-3000 Structural Biology."— Presentation transcript:

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

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

3 Bethesda, March 4 th 2009 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, ….

4 Bethesda, March 4 th 2009 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-3000partially build model Acta Cryst. D (2006) 62: 859-66. Times cited: 77 PDB deposits: >710

5 Bethesda, March 4 th 2009 Integrated system – HKL-3000 current 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.

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

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

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

9 Bethesda, March 4 th 2009 Integration HKL-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

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

11 Bethesda, March 4 th 2009 Completeness – overlaps Multi-crystal strategy

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

13 Bethesda, March 4 th 2009 Scaling Signal from Sulfur

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

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

16 Bethesda, March 4 th 2009 SAD or MAD phasing

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

18 Bethesda, March 4 th 2009 Substructure enantiomorph determination

19 Bethesda, March 4 th 2009 Double conformations

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

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

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

23 Bethesda, March 4 th 2009 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

24 Bethesda, March 4 th 2009 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

25 Bethesda, March 4 th 2009 TM1086 (APC4579)

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

27 Bethesda, March 4 th 2009 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. MCSG sequence from electron density map

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

29 Bethesda, March 4 th 2009 # of PDB deposits with given # of Mg ions 11273 2943 3210 4358 5-10274 11-5080 51-10033 >10152 # of Mg # of deposits

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

31 Bethesda, March 4 th 2009 Unknown ligands

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

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

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

35 Bethesda, March 4 th 2009 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 Resolution [Å] 4.0 Unique content in ASU [%] = = (1 – solvent content)/NCS factor No methods to interpret such maps Arp/wARP builds most of the model Atomicity based phasing e.g. ME Challenging projects 70 40 50 60 10 30 20 2.0 1.52.5 3.0 3.5

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

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

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