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In honor of Professor B.C. Wang receiving the 2008 Patterson Award

Direct Methods and Solvent Flattening. In honor of Professor B.C. Wang receiving the 2008 Patterson Award. Q. Hao, Y.X. Gu, C.D. Zheng & H.F. Fan. Direct methods breaking. the SIR/SAD phase ambiguity. Component Relationships ( 1965 ). Chinese Physics 1429-1435 (1965). SAD case:

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In honor of Professor B.C. Wang receiving the 2008 Patterson Award

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  1. Direct Methods and Solvent Flattening In honor of Professor B.C. Wang receiving the 2008 Patterson Award Q. Hao, Y.X. Gu, C.D. Zheng & H.F. Fan

  2. Direct methods breaking the SIR/SAD phase ambiguity

  3. Component Relationships (1965) Chinese Physics 1429-1435 (1965) SAD case: If the heavy-atom substructure is centrosymmetric, we know Ah and |Bh|, the question is to find the sign of Bh . SIR case: If the heavy-atom substructure is centrosymmetric, we know Bh and |Ah|, the question is to find the sign of Ah .

  4. P+ formula (1985) Acta Cryst. A40, 489-495 (1984) Acta Cryst. A40, 495-498 (1984) Acta Cryst. A41, 280-284 (1985) The phase problem reduces to a sign problem A combination of SIR/SAD bimodal distribution with the Cochran distribution incorporating with partial structure information

  5. Direct-method phasing of the 2Å experimental SAD data of the protein aPP Avian Pancreatic Polypeptide Space group: C2 Unit cell: a = 34.18, b = 32.92, c = 28.44Å; b = 105.3o Protein atoms in ASU: 301 Resolution limit: 2.0Å Anomalous scatterer: Hg (in centric arrangement) Wavelength: 1.542Å (Cu-Ka) Df” = 7.686 Locating heavy atoms & SAD phasing: direct methods Acta Cryst. (1990). A46, 935. Data courtesy ofProfessor Tom Blundell

  6. Much more complicated proteins have been solved by combining direct methods & solvent flattening: SAD phasing by OASIS + DM (Cowtan, CCP4)

  7. OASIS-2006 Institute of Physics Chinese Academy of Sciences Beijing 100080, P.R. China http://cryst.iphy.ac.cn http://www.ccp4.ac.uk/prerelease

  8. TTHA1634 from Thermus thermophilus HB8 Space group: P21212 Unit cell: a = 100.57, b = 109.10, c = 114.86Å Number of residues in the ASU: 1206 Resolution limit: 2.1Å Multiplicity: 29.2 Anomalous scatterer: S (22) X-ray wavelength: l = 1.542Å (Cu-Ka) Bijvoet ratio: <|DF|>/<F> = 0.55% Phasing method: A single run of OASIS2006 + DM (Cowtan) Model building: ARP/wARP Ribbon model plotted by PyMOL ARP/wARP found 1178 of the total 1206 residues, all docked into the sequence. Data courtesy ofProfessor Nobuhisa Watanabe Department of Biotechnology and Biomaterial Chemistry, Nagoya University, Japan

  9. What’s the low resolution limit ?

  10. SAD phasing at different resolutions TTHA1634 Cu-Ka data, <|DF|>/<F> ~ 0.55% Very good Marginally traceable 2.1Å 3.5Å 4.0Å Good 3.0Å Still informative Maps at 1s phased by a single run of OASIS + DM (Cowtan) plotted by PyMOL

  11. SAD phasing + Dual-space fragment extension by combination of OASIS, DM, RESOLVE & ARP/wARP

  12. Dual-space fragment extension Real-space fragment extension RESOLVE BUILD and/or ARP/wARP Reciprocal-space fragment extension OASIS + DM Partial model OK? No Yes End Partial structure

  13. Xylanase S-SAD Synchrotron l = 1.49Å Xylanase S-SAD Synchrotron l = 1.49Å Cycle 3 95% Cycle 0 42% Lysozyme S-SAD Cr-Ka Lysozyme S-SAD Cr-Ka 98% Cycle 6 52% Cycle 0 Azurin Cu-SAD Synchrotron l = 0.97Å Azurin Cu-SAD Synchrotron l = 0.97Å 99% Cycle 6 25% Cycle 0 Glucose isomerase S-SAD Cu-Ka Glucose isomerase S-SAD Cu-Ka Cycle 0 52% Cycle 4 97% Cr-Ka Se, S-SAD Alanine racemase Cr-Ka Se, S-SAD Alanine racemase 17% Cycle 0 97% Cycle 6 Ribbon models plotted by PyMOL Data courtesy ofProfessor N. Watanabe, Professor S. Hasnain, Dr. Z. Dauter and Dr. C. Yang

  14. Dual-space fragment extension without SAD/SIR information Direct-method aided MR-model completion

  15. ARP/wARP-OASIS-DM iteration 1UJZ Cycle 7 Cycle 5 Cycle 1 Cycle 3 201 residues all with side chains MR model Final model 46 residues 13 with side chains 215 residues ARP/wARP-DM iteration Cycle 2 Cycle 1 Ribbon models plotted by PyMOL

  16. Combining SOLVE/RESOLVE and OASIS + DM dealing with low resolution SIR/SAD data

  17. R-phycoerythrin SIR data from the native and the p-chloromercuriphenyl sulphonic acid derivative J.Mol.Biol.262721-731 (1996) Chinese Physics 16, 3022-3028 (2007) Maps plotted by PyMOL Space group: R3Unit cell: a = b = 189.8, c = 60.0Å;g = 120oNumber of residues in the ASU: 668 Resolution limit:2.8ÅReplacing atoms: Hg X-rays: Cu-Ka, λ = 1.542Å SOLVE/RESOLVE & OASIS + DM SOLVE/RESOLVE

  18. Tom70p Nature Structural & Molecular Biology 13, 589-593 (2006) Chinese Physics B 17, 1-9 (2008) Space group: P21Unit cell: a = 44.89, b = 168.8, c = 83.4Å;    β = 102.74oNumber of residues: 1086 Resolution limit: 3.3ÅMultiplicity: 3.3Anomalous scatterer: Se (24)X-rays: Synchrotron, λ = 0.9789Å, Δf" = 6.5Bijvoet ratio: <|ΔF|>/<|F|> = 4.3% Maps plotted by PyMOL SOLVE/RESOLVE & OASIS + DM SOLVE/RESOLVE & OASIS + DM SOLVE/RESOLVE SOLVE/RESOLVE

  19. Acknowledgements Professor Zhengjiong Lin Institute of Biophysics, Chinese Academy of Sciences, Beijing,China Drs Y. He1, D.Q. Yao1, J.W. Wang1, S. Huang1, J.R. Chen1, Q. Chen2, H. Li3, Prof. T. Jiang3, & Mr. T. Zhang1 1 Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, China 2 National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing, China 3 Institute of Biophysics, Chinese Academy of Sciences,Beijing China The project is supported by the Chinese Academy of Sciences and the 973 Project (Grant No 2002CB713801) of the Ministry of Science and Technology of China.

  20. Thank you!

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