Direct-method SAD phasing

Early attempts

1985 1965： Fan, H. F. Chinese Physics, 1429. 1966： Karle, J. Acta Cryst. 21, 273. 1970： Hazell, A. C. Nature 227, 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. 1985

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.

Direct phasing of the 3Å SAD data of the 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. protein core streptavidin

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

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

tetragonal hen egg-white lysozyme Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale J.-P. Ebel CEA-CNRS-UJF, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 01, France 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 OASIS + DM Éric Girard, Laurent Chantalat, Jean Vicat and Richard Kahn

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

Basis for structural diversity in homologous RNAs 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.

CrKα線を用いたSAD 法による位相決定 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。

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

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

OASIS-2004 application Xylanase Contoured at 1s 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

TT0570 OASIS-2004 application Space group: P21212 Unit cell: Data courtesy of Professor Isao Tanaka & Dr. Nobuhisa Watanabe Graduate School of Science, Hokkaido University, Japan 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 OASIS-2004 application

OASIS-2004 Features of

Better initial SAD phases

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

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

Cover figure of Acta Cryst. D60, Part 11 (2004) Se-SAD 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 phasing by OASIS-2004 + DM Data provided by Dr. S. J. Gamblin and Dr. B. Xiao Cover figure of Acta Cryst. D60, Part 11 (2004)

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

Comparison on cumulative phase errors sorted in descending order of Fobs 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

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

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

Br-SAD OASIS-2004 application Pepstatin-insenstive carboxylproteinase Contoured at 1s 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

Xe-SAD OASIS-2004 application Porcine Pancreatic Elastase Contoured at 1s 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

S-SAD OASIS-2004 application Lysozyme Contoured at 1s 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

302 residues found automatically 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

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

Examples Case study and

Azurin Cu-SAD Synchrotron l = 0.97Å Cycle 3 95% Azurin Cu-SAD Synchrotron l = 0.97Å Cycle 0 42% Xylanase S-SAD Synchrotron l = 1.49Å 99% Cycle 6 25% Cycle 0 Xylanase S-SAD Synchrotron l = 1.49Å Lysozyme S-SAD Cr-Ka 98% Cycle 6 52% Cycle 0 Lysozyme S-SAD Cr-Ka Glucose isomerase S-SAD Cu-Ka 17% Cycle 0 97% Cycle 6 Glucose isomerase S-SAD Cu-Ka Cr-Ka Se, S-SAD Alanine racemase Cycle 4 97% Cr-Ka Se, S-SAD Alanine racemase Cycle 0 52%

OASIS-2004 application Xylanase Contoured at 1s 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

Xylanase: average phase error decreased during dual-space iteration Phase error in degrees 70 60 50 40 30 20 80 10 Cycle 5 1 6 2 4 3 Xylanase: average phase error decreased during dual-space iteration Is OASIS necessary here? Is OASIS necessary here? Yes What would it be without using RESOLVE (build only)? - OASIS-2004 - DM - Partial model from RESOLVE BUILD or ARP/wARP No

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

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

Inside direct-method SAD phasing

SAD formulation

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

SAD formulation F + ?

P+ formula

Maximizing P(Djh) Þ Djh=b

Replacing Ehexp(ia) with mhEhexp(iabest) Þ

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

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

Thank you!