1. SAD phasing by OASIS-2004 SAD phasing by OASIS-2004

2. OASIS-2004 Freely available at http://cryst.iphy.ac.cn A program for direct-method phasing and reciprocal-space fragment extension with SAD/SIR data By J.W. Wang, Y.X. Gu*, C.D. Zheng, H.F. Fan* and Q. Hao * Corresponding authors

3. SAD data used in this presentation were kindly provided by Dr. Z. Dauter, Dr. S. J. Gamblin, Prof. S. Hasnain, Prof. I. Tanaka, Dr. N. Watanabe, Dr. M.S. Weiss, Dr. B. Xiao and Dr. C. Yang Calculations were done by Mr. D.Q. Yao, Dr. S. Huang and Dr. J.W. Wang Acknowledgements

4. Two examples on difficult SAD phasing Two examples on difficult SAD phasing

5. OASIS-2004 application Contoured at 1  Xylanase Space group: P2 1 Unit cell: a = 41.07, b = 67.14, c = 50.81Å  = 113.5 o Resolution limit: 1.75Å; Multiplicity: 15.9 Anomalous scatterer: S (5 ) X-rays:  synchrotron radiation  = 1.488Å;  f ” = 0.52 Bijvoet ratio: / = 0.56% Phasing: OASIS-2004 + DM (Cowtan) Model building: RESOLVE BUILD & ARP/wARP found 299 of the total 303 residues at the 6 th cycle of iteration Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA

6. TT0570 Data courtesy of Professor Isao Tanaka & Dr. Nobuhisa Watanabe Graduate School of Science, Hokkaido University, Japan Space group: P2 1 2 1 2 Unit cell: a = 100.2639 b = 108.9852 c = 114.6272Å Number of residues in the ASU: 1206 Resolution range: 50.00-2.01Å Multiplicity: 20.9 Anomalous scatterer: S (22) Wavelength: = 2.291Å;  f ” = 1.14 Bijvoet ratio: / = 1.16% Phasing: OASIS-2004 + DM (Cowtan) Model building: RESOLVE BUILD & ARP/wARP ARP/wARP found 1153 of the total 1206 residues after 2 cycles of iteration OASIS-2004 application

7. Features of OASIS-2004

8. 1. Better initial SAD phases

9. Bimodal distribution of SAD The phase of F” Phase information available in SAD Cochran distribution Peaked at any where from 0 to 2  Peaked at Sim distribution

10. Two different kinds of initial SAD phases P+P+ + P P Sim P Bimodal Sim-modified phases P + -modified phases P+P+ P Sim P Cochran

11. Se-SAD Histone Methyltransferase Set 7/9 Space group: P2 1 2 1 2 1 Unit cell: a = 66.09, b = 82.83, c = 116.15Å Number of residues in ASU: 560 Number of independent reflections: 16352 Resolution limit: 2.8Å Multiplicity: 3.8 Anomalous scatterer: Se(12)  = 0.9794Å;  f’ = -7.5,  f” = 6.5 Bijvoet ratio: / = 7.03% SAD phasing by OASIS-2004 + DM Data provided by Dr. S. J. Gamblin and Dr. B. Xiao

12. Comparison of the two kinds of initial phases using 4 typical reflections from the protein histone methyltransferase SET 7/9

13. Comparison on cumulative phase errors sorted in descending order of F obs Comparison on cumulative phase errors sorted in descending order of F obs 60.263.415000 58.461.913500 56.960.812000 62.365.216352 55.659.410500 54.158.79000 52.957. 87500 51.257.06000 50.056.54500 49.157.13000 45.857.11500 Errors of P + -modified phases ( o ) Number of reflections Errors of Sim-modified phases ( o )

14. 2. Inclusion and auto balance of the lack-of-closure error in the direct-method phasing formula 2. Inclusion and auto balance of the lack-of-closure error in the direct-method phasing formula

15. Automatic solution of protein structures OASIS-2004 DM Automatic solution of protein structures OASIS-2004 DM by a single run of RESOLVE (Build only) and/or ARP/wARP RESOLVE (Build only) and/or ARP/wARP + + + +

16. Br-SAD OASIS-2004 application Contoured at 1  Pepstatin-insenstive carboxylproteinase Space group: P6 2 Unit cell: a = b = 97.31, c = 82.94Å,  = 120 o Resolution limit: 1.8Å; Multiplicity: 5.45 Anomalous scatterer: Br (13) X-rays:  synchrotron radiation  = 0.9191Å;  f ” = 5.0 Bijvoet ratio: / = 7.06% Phasing: OASIS-2004 + DM (Cowtan) Model building: ARP/wARP found 358 of the total 372 residues Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA

17. Xe-SAD OASIS-2004 application Contoured at 1  Porcine Pancreatic Elastase Space group: P2 1 2 1 2 1 Unit cell: a = 50.2, b = 58.1, c = 74.3Å Resolution limit: 1.94Å; Total rotation range: 360 o Anomalous scatterer: Xe (1) X-rays:  synchrotron radiation  = 2.1Å;  f ” = 11.8 Bijvoet ratio: / = 5.76% Phasing: OASIS-2004 + DM (Cowtan) Model building: ARP/wARP found 236 of the total 240 residues Data courtesy of Dr. M. S. Weiss, EMBL Hamburg Outstation, c/o DESY, Germany

18. S-SAD OASIS-2004 application Contoured at 1  Lysozyme Space group: P4 3 2 1 2 Unit cell: a = 78.81, c = 36.80Å Atoms in the asymmetric unit: 1001 Resolution limit: 1.53Å; Multiplicity: 23 Anomalous scatterer: S (10), Cl (7) X-rays:  synchrotron radiation  = 1.54Å;  f ” = 0.56, 0.70 Bijvoet ratio: / = 1.55% Phasing: OASIS-2004 + DM (Cowtan) Model building: ARP/wARP found 128 of the total 129 residues Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA

19. Cu-K  Fe-SAD 302 residues found automatically YfbpA Space group: P2 1 2 1 2 1 Unit cell: a = 42.792, b = 54.134, c = 115.222Å Resolution range: 57.74 – 1.42Å Anomalous scatterer: Fe (1) Wavelength: 1.542Å  f ” = 3.20 / ~ 1.4% Phased by: OASIS + DM (Cowtan) Automatic model building by: ARP/wARP Data provided by Dr. Cheng Yang Rigaku/MSC, USA OASIS-2004 application

20. 3. Dual-space fragment extension Partial model Partial model Partial model Partial model No Yes Reciprocal-space fragment extension by OASIS-2004 + DM Reciprocal-space fragment extension by OASIS-2004 + DM Real-space fragment extension by RESOLVE BUILD and/or ARP/wARP Real-space fragment extension by RESOLVE BUILD and/or ARP/wARP OK? End

21. 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%

22. Case study 1. Azurin 2. Xylanase 1. Azurin 2. Xylanase

23. Contoured at 1  Space group: P4 1 22 Unit cell: a = b = 52.65, c = 100.63Å Resolution limit: 1.9Å; Multiplicity: 10.0 Anomalous scatterer: Cu (1) X-rays: Synchrotron radiation  =  0.97Å;  f” = 2.206; / = 1.44% Phasing: OASIS-2004 + DM (Cowtan) Model building: RESOLVE BUILD and ARP/wARP 116 of 129 residues found automatically Data courtesy of Professor S. Hasnain Azurin

24. 70 60 50 40 30 80  OASIS-2004  DM  Partial model from RESOLVE BUILD or ARP/wARP Cycle 01 2 4 3 Phase error in degrees OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration Azurin copper-SAD phasing Synchrotron radiation = 0.97Å, / = 1.44% Phase error in degrees What would it be without using RESOLVE (build only)? Is OASIS necessary here? Is OASIS necessary here?

25. OASIS-DM-ARP/wARP Iteration Azurin copper-SAD phasing Synchrotron radiation = 0.97Å, / = 1.44% OASIS-DM-ARP/wARP Iteration Azurin copper-SAD phasing Synchrotron radiation = 0.97Å, / = 1.44% Phase error in degrees 0 10 2 4 8 6 Cycle 70 60 50 40 30 80 70 60 50 40 30 80  OASIS-2004  DM  Partial model from ARP/wARP

26. Cycle 0 (ARP/wARP) Cycle 4 (ARP/wARP) Cycle 6 (ARP/wARP) Cycle 8 (ARP/wARP) OASIS-DM-ARP/wARP Iteration Cycle 0 (RESOLVE BUILD) Cycle 1 (ARP/wARP) Cycle 2 (ARP/wARP) Cycle 3 (ARP/wARP) OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration Cycle 10 (ARP/wARP) Cycle 4 (ARP/wARP)

27. Cycle 0 Cycle 2 Cycle 4 Improvement on electron-density map and automatic model building Improvement on electron-density map and automatic model building Cycle 0 Cycle 2 Cycle 4

28. Xylanase Contoured at 1  Space group: P2 1 Unit cell: a = 41.07, b = 67.14, c = 50.81Å  = 113.5 o Resolution limit: 1.75Å; Multiplicity: 15.9 Anomalous scatterer: S (5 ) X-rays:  synchrotron radiation  = 1.488Å;  f ” = 0.52 Bijvoet ratio: / = 0.56% Phasing: OASIS-2004 + DM (Cowtan) Model building: RESOLVE BUILD & ARP/wARP found 299 of the total 303 residues at the 6 th cycle of iteration Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA

29.  OASIS-2004  DM  Partial model from RESOLVE BUILD or ARP/wARP Phase error in degrees 70 60 50 40 30 20 80 10 Cycle 0 5 1 6 2 4 3 OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration Xylanase sulfur-SAD phasing Synchrotron radiation = 1.49Å, / = 0.56% Is OASIS necessary here? What would it be without using RESOLVE (build only)? Is OASIS necessary here?

30. OASIS-DM-ARP/wARP Iteration Xylanase sulfur-SAD phasing Synchrotron radiation = 1.49Å, / = 0.56% OASIS-DM-ARP/wARP Iteration Xylanase sulfur-SAD phasing Synchrotron radiation = 1.49Å, / = 0.56% 70 60 50 40 30 20 80 0 10 2 12 4 8 6 14 16 Phase error in degrees Cycle  OASIS-2004  DM  Partial model from ARP/wARP

31. Cycle 14 (ARP/wARP) Cycle 16 (ARP/wARP) Cycle 17 (ARP/wARP) Cycle 0 (ARP/wARP) Cycle 0 (RESOLVE BUILD) Cycle 3 (ARP/wARP) Cycle 5 (ARP/wARP) Cycle 6 (ARP/wARP) OASIS-DM-ARP/wARP Iteration OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration

32. Cycle 0 Cycle 3 Cycle 6 Improvement on electron-density map and automatic model building Improvement on electron-density map and automatic model building

33. Conclusion 1. OASIS is essential for initial SAD phasing and for initiating a successful dual-space fragment extension; 2. Starting models from RESOLVE (build only) lead much faster to the final solution; 3. ARP/wARP models with dummy atoms only may lead to nearly the complete structure after sufficient cycles of iteration.

34. Thank you!