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SAD phasing by OASIS-2004

SAD phasing by OASIS-2004. OASIS-2004. 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. Freely available at http://cryst.iphy.ac.cn. Acknowledgements.

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SAD phasing by OASIS-2004

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  1. SAD phasing by OASIS-2004

  2. OASIS-2004 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 Freely available athttp://cryst.iphy.ac.cn

  3. Acknowledgements 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

  4. Two examples on difficult SAD phasing

  5. OASIS-2004 application Xylanase Space group: P21 Unit cell: a = 41.07, b = 67.14, c = 50.81Å b = 113.5o Resolution limit: 1.75Å; Multiplicity: 15.9 Anomalous scatterer: S(5 ) X-rays:synchrotron radiation l = 1.488Å; D f ” = 0.52 Bijvoet ratio:<|DF |>/<F > = 0.56% Phasing: OASIS-2004 + DM (Cowtan) Model building: RESOLVE BUILD & ARP/wARP found 299 of the total 303 residues at the 6th cycle of iteration Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA Contoured at 1s

  6. TT0570 OASIS-2004 application Space group: P21212 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: l = 2.291Å; Df ” = 1.14 Bijvoet ratio: <|DF|>/<F> = 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 Data courtesy ofProfessor Isao Tanaka & Dr. Nobuhisa Watanabe Graduate School of Science, Hokkaido University, Japan

  7. Features of OASIS-2004

  8. Better initial SAD phases

  9. Phase information available in SAD Bimodal distribution of SAD Cochran distribution Sim distribution Peaked at Peaked at any where from 0 to2p The phase of F”

  10. Sim-modified phases PSim PBimodal PSim PCochran P+ P + P+ P+-modified phases Two different kinds of initial SAD phases

  11. Histone Methyltransferase Set 7/9 Space group: P212121 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) l = 0.9794Å; Df’ = -7.5, Df” = 6.5 Bijvoet ratio: <|DF|>/<F> = 7.03% SAD phasingbyOASIS-2004 + DM Data provided by Dr. S. J. Gamblin and Dr. B. Xiao Se-SAD

  12. Comparisonof 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 descendingorder ofFobs Number of reflections Errors of Sim-modified phases (o) Errors of P+-modified phases (o) 1500 57.1 45.8 3000 57.1 49.1 4500 56.5 50.0 6000 57.0 51.2 7500 57. 8 52.9 9000 58.7 54.1 10500 59.4 55.6 12000 60.8 56.9 13500 61.9 58.4 15000 63.4 60.2 16352 65.2 62.3

  14. 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 by a single run of RESOLVE (Build only) and/or ARP/wARP + +

  16. OASIS-2004 application Pepstatin-insenstive carboxylproteinase Space group: P62 Unit cell: a = b = 97.31, c = 82.94Å, g = 120o Resolution limit: 1.8Å; Multiplicity: 5.45 Anomalous scatterer: Br (13) X-rays:synchrotron radiation l = 0.9191Å; D f ” = 5.0 Bijvoet ratio: <|D F |>/<F > = 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 Br-SAD Contoured at 1s

  17. OASIS-2004 application Porcine Pancreatic Elastase Space group: P212121 Unit cell: a = 50.2, b = 58.1, c = 74.3Å Resolution limit: 1.94Å; Total rotation range: 360o Anomalous scatterer: Xe (1) X-rays:synchrotron radiation l = 2.1Å; D f ” = 11.8 Bijvoet ratio: <|D F |>/<F > = 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 Xe-SAD Contoured at 1s

  18. OASIS-2004 application Lysozyme Space group: P43212 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 l = 1.54Å; D f ” = 0.56, 0.70 Bijvoet ratio: <|D F |>/<F > = 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 S-SAD Contoured at 1s

  19. OASIS-2004 application YfbpA Space group: P212121 Unit cell: a = 42.792, b = 54.134, c = 115.222Å Resolution range: 57.74 – 1.42Å Anomalous scatterer: Fe (1) Wavelength: 1.542Å Df ” = 3.20 <|DF|>/<F> ~ 1.4% Phased by: OASIS + DM (Cowtan) Automatic model building by: ARP/wARP Data provided by Dr. Cheng Yang Rigaku/MSC, USA Cu-Ka Fe-SAD 302 residues found automatically

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

  21. 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

  22. Case study • Azurin • Xylanase

  23. Azurin Space group: P4122 Unit cell: a = b = 52.65, c = 100.63Å Resolution limit: 1.9Å; Multiplicity: 10.0 Anomalous scatterer: Cu (1) X-rays: Synchrotron radiationl =0.97Å; Df” = 2.206; <|DF|>/<F> = 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 Contoured at 1s

  24. OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration Azurin copper-SAD phasing Synchrotron radiation l = 0.97Å, <DF>/<F> = 1.44% 80 70 60 Phase error in degrees 50 - OASIS-2004 - DM - Partial model from RESOLVE BUILD or ARP/wARP 40 30 4 3 0 1 2 Cycle Is OASIS necessary here? Is OASIS necessary here? Phase error in degrees What would it be without using RESOLVE (build only)?

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

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

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

  28. Xylanase Space group: P21 Unit cell: a = 41.07, b = 67.14, c = 50.81Å b = 113.5o Resolution limit: 1.75Å; Multiplicity: 15.9 Anomalous scatterer: S(5 ) X-rays:synchrotron radiation l = 1.488Å; D f ” = 0.52 Bijvoet ratio:<|DF |>/<F > = 0.56% Phasing: OASIS-2004 + DM (Cowtan) Model building: RESOLVE BUILD & ARP/wARP found 299 of the total 303 residues at the 6th cycle of iteration Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA Contoured at 1s

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

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

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

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

  33. Conclusion • 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 maylead to nearly the complete structure after • sufficient cycles of iteration.

  34. Thank you!

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