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Direct-method SAD phasing

Explore the evolution of direct-method phasing in protein crystallography from early attempts to breakthroughs in 3D structure determination. Discover key studies, software tools, and techniques for successful SAD phasing in macromolecular structures.

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Direct-method SAD phasing

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  1. Direct-method SAD phasing

  2. Early attempts

  3. 1985 1965： Fan, H. F. Chinese Physics, 1429. 1966： Karle, J. Acta Cryst. 21, 273. 1970： Hazell, A. C. Nature227, 269. 1973: Sikka, S. K. Acta Cryst. A29, 211. 1978: Heinerman, J. J. L., Krabbendam, H., Kroon, J. & Spek, A. L. Acta Cryst. A34, 447. 1982： Hauptman, H. A. Acta Cryst. A38, 632. 1983: Giacovazzo, C. Acta Cryst. A39, 585. 1985: Fan, H. F. and Gu, Y. X. Acta Cryst. A41, 280. 1993: Kyriakidis, C. E., Peschar, R. and Schenk, H. Acta Cryst. A49, 557. 1998: Fan, H. F. et al. in Direct Methods for Solving Macromolecular Structures, Kluwer Academic Publishes, pp. 479-485.

  4. 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, Zn Wavelength: 1.542Å (Cu-Ka) Df” = 7.686, 0.678 Phasing: direct methods Acta Cryst. (1990). A46, 935.

  5. Direct phasing of the 3Å SAD data of the protein core streptavidin Space group: I222 Unit cell: a=95.27; b=105.41, c= 47.56Å Protein atoms in ASU: 1836 Resolution limit: 3.0Å Anomalous scatterer: Se l = 0.9795Å; Df” = 3.663 Phasing: direct methods + solvent flattening + non-crystallographic symmetry averaging Acta Cryst. (1995). D51, 342.

  6. OASIS (The first edition, 2000) A computer program for breaking the phase ambiguity in One-wavelength Anomalous Scattering or Single Isomorphous Replacement (Substitution) protein data. http://www.ccp4.ac.uk/dist/html/oasis.html

  7. Mlphare + dm Oasis + dm Acta Cryst. (1999). D55, 1620-1622. The first example of solving an unknown protein by direct-method phasing of 2.1Å SAD data Rusticyanin, MW: 16.8 kDa; SG: P21; a=32.43, b=60.68, c=38.01Å ; b=107.82o ; Anomalous scatterer: Cu

  8. Acta Cryst. D58, 1-9 (2002). Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozyme Éric Girard, Laurent Chantalat, Jean Vicat and Richard Kahn Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale J.-P. Ebel CEA-CNRS-UJF, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 01, France OASIS + DM OASIS

  9. J. Mol. Biol.348, 951–959 (2005) Crystal Structures of Fms1 and its Complex with Spermine Reveal Substrate Specificity Qingqiu Huang1, Qun Liu1 and Quan Hao1,2 1MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University Ithaca, NY 14853-8001, USA 2Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China

  10. Science, Vol. 306, Issue 5693, 104-107 (2004) Basis for structural diversity in homologous RNAs Andrey S. Krasilnikov*, Yinghua Xiao*, Tao Pan†, and Alfonso Mondragón* * Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA † Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA For SAD phasing, the positions of the first 3 Ba+2 sites were identified using SOLVE, 6 more Ba+2 sites were identified and added during heavy atom refinement with SHARP. The phase ambiguity in SAD phasing was partially resolved using OASIS and solvent flattening with SOLOMON as implemented in SHARP. Further improvement of the phases was achieved by doing iterative cycles of phase refinement incorporating phase information from partially built models followed by solvent flattening.

  11. 構造生物 Vol.10 No.1 2004 年2 月発行 CrKα線を用いたSAD 法による位相決定 理学電機(株) 山野昭人、佐藤貴久、長谷川智一 初期位相の決定はSHARP やOASIS が成績が良い。MLPHARE でも可能との事だが、筆者の使用法の問題だと思われるが、これまで成功した経 験がない。 図2 はサウマチンの解析例である。1 フレームを0.5度振動、1 分露光で180 度分測定したイメージをHKL2000 で処理。15-3Åのデータを用いてSHELX97 を実行。8 個のS-S の位置と1 個の硫黄原子の位置全てが決定できた。これらの座標を基にOASIS を実行。初期位相を決定した。DM およびSOLOMON に より位相を改良した。

  12. OASIS-2004 A direct-method program for ab initio phasing and reciprocal-space fragment extension with SAD/SIR data Institute of Physics, CAS, Beijing, P.R. China http://cryst.iphy.ac.cn

  13. Difficult SAD phasing • SAD phasing at Bijvoet ratio ~ 0.56% • An originally unknown protein with 1206 residues in the ASU solved automatically using Cr-Ka sulfur-SAD data

  14. 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

  15. 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

  16. Features of OASIS-2004

  17. Better initial SAD phases

  18. 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”

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

  20. 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 Cover figure of Acta Cryst. D60, Part 11 (2004)

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

  22. 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

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

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

  25. 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

  26. 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

  27. 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

  28. 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

  29. 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

  30. Examples Case study and

  31. 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

  32. 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

  33. Xylanase: average phase error decreased during dual-space iteration 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? Yes What would it be without using RESOLVE (build only)? No

  34. 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

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

  36. Inside direct-method SAD phasing

  37. SAD formulation

  38. SAD formulation F” - F” F + F + F’ F*- F o

  39. F + ? F + SAD formulation

  40. P+ formula

  41. Maximizing P(Djh) Þ Djh=b

  42. ReplacingEhexp(ia)with mhEhexp(iabest) Þ

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

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

  45. Thank you!

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