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

2. the SIR/SAD phase ambiguity Direct methods breaking

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

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;  = 105.3 o Protein atoms in ASU: 301 Resolution limit: 2.0Å Anomalous scatterer: Hg (in centric arrangement) Wavelength: 1.542Å (Cu-K  )  f” = 7.686 Locating heavy atoms & SAD phasing: direct methods Acta Cryst. (1990). A46, 935. Avian Pancreatic Polypeptide Space group: C2 Unit cell: a = 34.18, b = 32.92, c = 28.44Å;  = 105.3 o Protein atoms in ASU: 301 Resolution limit: 2.0Å Anomalous scatterer: Hg (in centric arrangement) Wavelength: 1.542Å (Cu-K  )  f” = 7.686 Locating heavy atoms & SAD phasing: direct methods Acta Cryst. (1990). A46, 935. Data courtesy of Professor Tom Blundell

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

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

8. TTHA1634 from Thermus thermophilus HB8 Data courtesy of Professor Nobuhisa Watanabe Department of Biotechnology and Biomaterial Chemistry, Nagoya University, Japan Space group: P2 1 2 1 2 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: = 1.542Å (Cu-K  ) Bijvoet ratio: / = 0.55% Phasing method: A single run of OASIS2006 + DM (Cowtan) Model building: ARP/wARP ARP/wARP found 1178 of the total 1206 residues, all docked into the sequence. Ribbon model plotted by PyMOL

9. What’s the low resolution limit ? What’s the low resolution limit ?

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

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

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

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

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

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

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

17. R-phycoerythrin SIR data from the native and the p-chloromercuriphenyl sulphonic acid derivative Space group: R3 Unit cell: a = b = 189.8, c = 60.0Å;  = 120 o Number of residues in the ASU: 668 Resolution limit: 2.8Å Replacing atoms: Hg X-rays: Cu-K , λ = 1.542Å J.Mol.Biol. 262 721-731 (1996) Chinese Physics 16, 3022-3028 (2007) SOLVE/RESOLVE & OASIS + DM Maps plotted by PyMOL

18. SOLVE/RESOLVE & OASIS + DM SOLVE/RESOLVE SOLVE/RESOLVE & OASIS + DM Tom70p Space group: P2 1 Unit cell: a = 44.89, b = 168.8, c = 83.4Å; β = 102.74 o Number of residues: 1086 Resolution limit: 3.3Å Multiplicity: 3.3 Anomalous scatterer: Se (24) X-rays: Synchrotron, λ = 0.9789Å, Δf" = 6.5 Bijvoet ratio: / = 4.3% Nature Structural & Molecular Biology 13, 589-593 (2006) Chinese Physics B 17, 1-9 (2008) Maps plotted by PyMOL

19. Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Acknowledgements Professor Zhengjiong Lin 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 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 Drs Y. He 1, D.Q. Yao 1, J.W. Wang 1, S. Huang 1, J.R. Chen 1, Q. Chen 2, H. Li 3, Prof. T. Jiang 3, & Mr. T. Zhang 1 Drs Y. He 1, D.Q. Yao 1, J.W. Wang 1, S. Huang 1, J.R. Chen 1, Q. Chen 2, H. Li 3, Prof. T. Jiang 3, & Mr. T. Zhang 1 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!