1. H.F. Fan 1, Y.X. Gu 1, F. Jiang 1,2 & B.D. Sha 3 1 Institute of Physics, CAS, Beijing, China 2 Tsinghua University, Beijing, China 3 University of Alabama at Birmingham, USA H.F. Fan 1, Y.X. Gu 1, F. Jiang 1,2 & B.D. Sha 3 1 Institute of Physics, CAS, Beijing, China 2 Tsinghua University, Beijing, China 3 University of Alabama at Birmingham, USA Resolving OAS phase ambiguity and improving MAD phases Resolving OAS phase ambiguity and improving MAD phases Direct-method phasing of anomalous diffraction Direct-method phasing of anomalous diffraction Why direct method ? Is it important?

2. Synchrotron Radiation + Synchrotron Radiation + Selenomethionine enrichment + Selenomethionine enrichment + MAD can be improved by making use of Direct Methods can be improved by making use of Direct Methods The main phasing technique in structural genomics The main phasing technique in structural genomics Systematic study of protein structures based on genome sequencing for better understanding of life process and improving human health Systematic study of protein structures based on genome sequencing for better understanding of life process and improving human health High intensity and tunable wavelength High intensity and tunable wavelength Replacing S atoms with Se atoms Replacing S atoms with Se atoms Multi-wavelength Anomalous Diffraction Multi-wavelength Anomalous Diffraction Ethan A Merritt ©1996-2000 merritt@u.washington.edu Biomolecular Structure Center at UW

3. 1. Direct-method phasing of One-wavelength Anomalous Scattering (OAS) data 2. MAD data treated by direct methods as n-sets of OAS data 1. Direct-method phasing of One-wavelength Anomalous Scattering (OAS) data 2. MAD data treated by direct methods as n-sets of OAS data

4. OAS (SAD) formulation F + F” F’ F o - F” F + F* 

5. OAS formulation F + ?

6. F’ SIRAS (SIR+OAS) FNFN FNFN FRFR FDFD FDFD

7. F’ MAD

8. OAS+Sim + solvent flattening ISAS F’

9.   ”” Difference of the Bijvoet pair PP  The phase of F” Breaking the OAS phase ambiguity The phase of  ”+   ”  ’        ”+   ”  ’       The phase of the reflection h

11. Maximizing P(  h )   h = 

12. Replacing E h exp(i  ) with m h E h exp(i  best ) 

13. Fan, H.F. & Gu, Y.X., Acta Cryst. A41, 280-284 (1985)

14. Fan, H.F. & Gu, Y.X., Acta Cryst. A41, 280-284 (1985)

15. Direct-method OAS phasing OAS data P + (  h ) = 1/2 End Calculate P + (  h )  h  ’ h,   h | Calculate m h and  h,best

16. The first example of solving an unknown protein by direct- method phasing of the 2.1 Å OAS data Rusticyanin, MW: 16.8 kDa; SG: P2 1 ; a=32.43, b=60.68, c=38.01Å ;  =107.82 o ; Anomalous scatterer: Cu Rusticyanin, MW: 16.8 kDa; SG: P2 1 ; a=32.43, b=60.68, c=38.01Å ;  =107.82 o ; Anomalous scatterer: Cu Mlphare + dm Oasis + dm OAS distribution Sim distribution Cochran distribution Solvent flattening OAS distribution Sim distribution Solvent flattening

17. MAD phasing Direct-method OAS phasing MAD phasing Direct-method OAS phasing Ompdc Pure Comparison of OAS and MAD phasing (data from Dr. S. Ealick)

18. OASIS (CCP4: Supported Program) DESCRIPTION OASIS is a computer program for breaking phase ambiguity in One-wavelength Anomalous Scattering or Single Isomorphous Replacement (Substitution) protein data. The phase problem is reduced to a sign problem once the anomalous-scatterer or the replacing-heavy-atom sites are located. OASIS applies a direct method procedure to break the phase ambiguity intrinsic to OAS or SIR data. REFERENCES Fan, H. F. and Gu, Y. X. (1985) Combining direct methods with isomorphous replacement or anomalous scattering data III. The incorporation of partial structure information, Acta Cryst. A41, 280-284. Fan H. F., Hao, Q., Gu, Y. X., Qian, J. Z., Zheng, C. D. and Ke, H. (1990) Combining direct methods with isomorphous replacement or anomalous scattering data VII. Ab initio phasing of the OAS data from a small protein, Acta Cryst. A46, 935-939. Y. -D. Liu, I. Harvey, Y. -X. Gu, C. -D. Zheng, Y. -Z. He, H. -F. Fan, S. S. Hasnain and Q. Hao (1999) Is single-wavelength anomalous scattering sufficient for solving phases? A comparison of different methods for a 2.1 A structure solution, Acta Cryst. D55, 1620- 1622. AUTHORS Q. Hao (1, 2), Y. X. Gu, C. D. Zheng & H. F. Fan (2) (1) School of Applied Sciences, De Montfort University, Leicester LE1 9BH, England. (2) Institute of Physics, Chinese Academy of Sciences, Beijing 100080, P. R. China. Email: qhao@dmu.ac.uk or fan@aphy.iphy.ac.cn

19. 1. Direct-method phasing of One-wavelength Anomalous Scattering (OAS) data 2. MAD data treated by direct methods as n-sets of OAS data 1. Direct-method phasing of One-wavelength Anomalous Scattering (OAS) data 2. MAD data treated by direct methods as n-sets of OAS data

20. Direct-method aided MAD phasing MAD data Conventional MAD phasing MAD phases with figure of merit > 0.9 n sets of OAS data Direct-method OAS phasing Phase combination End MAD phases

21. Phase combination 1 2 OAS phases Set n OAS phases Set 1 Combine n sets of OAS phases Combine OAS and MAD phases MAD phases

22. Direct-method aided MAD phasing (Sample: Adenosine Kinase) Space group: P2 1 2 1 2 Unit cell: a = 64.528, b = 109.841, c=49.231Å Non-H atoms in a.s.u.: 2,695 Number of Se sites in a.s.u:6 Wavelength (Å): 0.9790 0.9787 0.9500 Resolution: 20  2.5 Å Unique reflections: 12525

23. Direct-method aided MAD phasing (Sample: Adenosine Kinase) Spacegroup P2 1 2 1 2 Unit cella = 64.528, b = 109.841, c =49.231Å Non-H atoms in a.s.u. 2,695 (3,093 including water molecules) Number of Se sites in a.s.u6 SourceX-12C at National Synchrotron Light Source Data collection protocolinverse beam Wavelength (Å)0.9790 0.9787 0.9500 f’ (in electrons)  7.381  6.830  2.739 f" (in electrons) 3.840 3.838 3.605 Resolution 20  2.5 Å Unique reflections12525 Completeness 99.0%

24. MAD DMAD Direct-method aided MAD phasing (data from Dr. S. Ealick) Direct-method aided MAD phasing (data from Dr. S. Ealick) MAD information Sim distribution Cochran distribution Solvent flattening MAD information Sim distribution Solvent flattening

25. Direct-method aided MAD phasing Sample: yeast Hsp40 protein Sis1 (171  352) Space group: P4 1 2 1 2 Unit cell: a = 73.63, c =80.76Å Independent non-H atoms: 1380 Number of Se sites in a.s.u: 1 Wavelength (Å): 1.0688 0.9798 0.9794 0.9253 Resolution: 30  3.0 Å Unique reflections: 4590 Space group: P4 1 2 1 2 Unit cell: a = 73.63, c =80.76Å Independent non-H atoms: 1380 Number of Se sites in a.s.u: 1 Wavelength (Å): 1.0688 0.9798 0.9794 0.9253 Resolution: 30  3.0 Å Unique reflections: 4590

26. Direct-method aided MAD phasing Sample: yeast Hsp40 protein Sis1 (171  352) Spacegroup P4 1 2 1 2 Unit cella = 73.63, c =80.76Å Non-H atoms in a.s.u. 1380 including water molecules Number of Se sites in a.s.u1 Source SSRL station 7-1 Energy bands (eV)11600 12654 12659 13400 Wavelength (Å) 1.0688 0.9798 0.9794 0.9253 f’ (in electrons)  3.0191  9.5295  9.3527  2.7144 f” (in electrons) 0.9711 5.1236 4.8013 2.7489 Resolution 30  3.0 Å Unique reflections4591 Completeness 88.8 %

27. Number of MAD (4w) DMAD reflections + dm (CCP4) (2w) (4w) Number of MAD (4w) DMAD reflections + dm (CCP4) (2w) (4w) Phase errors of different groups cumulated in descending order of Fobs Phase errors of different groups cumulated in descending order of Fobs 500 19.36 18.28 16.02 1000 23.81 22.57 21.16 1500 26.04 25.73 23.47 2000 28.13 27.45 25.43 2500 30.20 29.78 27.20 3000 32.35 31.50 28.99 3500 34.11 33.22 30.69 4000 35.99 34.94 32.35 4500 37.40 36.36 33.98

28. Number of FOM MAD FOM DMAD FOM DMAD reflections min. +dm (CCP4) min. (2w) min. (4w) 500 1.00 16.58 0.97 13.54 1.00 13.79 500 0.97 23.29 0.93 22.87 0.97 21.58 500 0.95 32.73 0.87 *32.84 0.94 26.26 500 0.93 35.98 0.81 *38.48 0.92 *37.59 500 0.89 41.98 0.72 41.36 0.89 38.71 500 0.84 45.71 0.61 *49.22 0.85 38.23 500 0.75 53.68 0.49 *57.46 0.78 44.94 500 0.56 64.59 0.38 *66.09 0.67 48.75 500 0.14 80.26 0.26 72.19 0.50 61.91 Phase error vs. figure of merit

29. 4w-MAD 2w-DMAD Direct-method aided MAD phasing (yeast Hsp40 protein Sis1: 171  352)

30. 2w-DMAD 4w-MAD Direct-method aided MAD phasing (yeast Hsp40 protein Sis1: 171  352)

31. 2w-DMAD 4w-MAD Direct-method aided MAD phasing (yeast Hsp40 protein Sis1: 171  352)

32. The above procedures can be applied also to SIR or MIR data The above procedures can be applied also to SIR or MIR data

33. Direct phasing of the 2 Å SIR data of aPP with the replacing atoms (Hg) in centrosymmetric arrangement Space group: C2 Unit cell: a=34.18, b=32.92, c=28.22Å;  =105.3 o with the replacing atoms (Hg) in centrosymmetric arrangement Space group: C2 Unit cell: a=34.18, b=32.92, c=28.22Å;  =105.3 o Liu, Y.D., Gu, Y.X,, Zheng, C.D, & Fan, H.F., Acta Cryst. D55, 846-848 (1999) Liu, Y.D., Gu, Y.X,, Zheng, C.D, & Fan, H.F., Acta Cryst. D55, 846-848 (1999) SIR D-SIR

34. OASIS (CCP4 supported) D-MAD Multi-solution random-starting phasing Cluster analysis |  iteration D-SIRAS, D-MIR & D-MR Laue DMAD Incorporation of solution scattering ……

35. Acknowledgements Zheng, C. D., Liu, Y. D. (Institute of Physics, Beijing, China) Hao, Q. (MacCHESS, Cornell University, USA) Project 973: G1999075604 (Department of Science & Technology, China) Acknowledgements Zheng, C. D., Liu, Y. D. (Institute of Physics, Beijing, China) Hao, Q. (MacCHESS, Cornell University, USA) Project 973: G1999075604 (Department of Science & Technology, China)

36. Thank you !