International Conference on Structural Genomics 2006 Hands-on Workshop III International Conference on Structural Genomics 2006 Hands-on Workshop III Automated Structural Determination by X-ray Crystallography Automated Structural Determination by X-ray Crystallography Yokohama, Japan October 19-20, 2006

OASIS-2006 Institute of Physics Chinese Academy of Sciences Beijing , P.R. China Institute of Physics Chinese Academy of Sciences Beijing , P.R. China

OASIS (2000) in CCP4 OASIS-2004 on the Web OASIS-2006 on the Web soon

OASIS-2006 GUI for CCP4i OASIS-2006 GUI for CCP4i

1. Direct-method 2. Reciprocal-space 1. Direct-method 2. Reciprocal-space Functions of OASIS SAD/SIR Phasing Fragment Extension Dual-space by combining with DM, RESOLVE and ARP/wARP by combining with DM, RESOLVE and ARP/wARP

Why direct methods? For better initial SAD phases! For better initial SAD phases!

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

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

Comparison of 4 typical reflections from the protein histone methyltransferase SET 7/9

Comparison of cumulative phase errors in descending order of F obs Comparison of cumulative phase errors in descending order of F obs Errors of P + -modified phases ( o ) Number of reflections Errors of Sim-modified phases ( o ) Histone methyltransferase SET 7/9

Why dual-space? For less systematic errors! For less systematic errors!

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

OASIS Applications

Ab initio SAD phasing with weak anomalous signal with weak anomalous signal

SHARP-SOLOMON-ARP/wARP Xylanase Space group: P2 1 Unit cell: a = 41.07, b = 67.14, c = 50.81Å  = o Number of residues in the ASU: 303 Resolution limit: 1.75Å; Multiplicity: 15.9 Anomalous scatterer: S (5 ) X-rays:  synchrotron radiation  = 1.488Å;  f ” = 0.52 Bijvoet ratio: / = 0.56% Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA OASIS-DM-RESOLVE BUILD cycle 0 25% OASIS-DM-ARP/wARP cycle 6 99% SHARP-DM-RESOLVE BUILD BP3-DM-RESOLVE BUILD

2.1Å 3.5Å 4.0Å SAD phasing at different resolutions TT0570 Cu-K  data, / ~ 0.55% SAD phasing at different resolutions TT0570 Cu-K  data, / ~ 0.55%

OASIS + DM SOLVE/RESOLVE results improved by

SOLVE/RESOLVE SOLVE/RESOLVE + OASIS-DM-RESOLVE (BUILD) Dual-space fragment extension based on SOLVE/RESOLVE results 1LIA (d14) 2.8Å SIR data

SOLVE/RESOLVE map Sigma_A map based on a model manually built from the SOLVE/RESOLVE map OASIS-DM map based on the same model Dual-space fragment extension based on SOLVE/RESOLVE results 2GW1 3.3Å SAD data

Dual-space fragment extension based on SOLVE/RESOLVE results 2GW1 3.3Å SAD data Sigma_A map based on a model manually built from the SOLVE/RESOLVE map SOLVE/RESOLVE mapOASIS-DM map based on the same model

Molecular Replacement Fragment extension based on

Fragment extension based on molecular replacement Final model acidic phospholipase A2 124 residues MR model 60 residues 48 residues ARP/wARP-OASIS-DM iteration Cycle 1 Cycle 2Cycle 3 ARP/wARP-DM iteration Cycle 9 Cycle 11 Cycle 13 ARP/wARP-DM iteration

A difficult case: 1UJZ Space group: I 222 Unit cell: a=62.88, b=74.55, c=120.44Å Number of residues in ASU: 215 molecule A: 87 residues molecule B: 128 residues Number of residues in the starting model: 46 (all in Mol. A) Resolution range: – 2.10Å Number of reflections: Space group: I 222 Unit cell: a=62.88, b=74.55, c=120.44Å Number of residues in ASU: 215 molecule A: 87 residues molecule B: 128 residues Number of residues in the starting model: 46 (all in Mol. A) Resolution range: – 2.10Å Number of reflections: 16460

Fragment Extension Dual-space without SAD/SIR information

Partial structure Partial structure Density modification by DM Density modification by DM No MR model MR model Yes End Model rebuild by RESOLVE BUILD or ARP/wARP Model rebuild by RESOLVE BUILD or ARP/wARP OK? Phase improvement by OASIS Phase improvement by OASIS

P + > 0.5  ”  model P + < 0.5  ”   model    ~   ~   ”” 

Range of phase error in degrees Cycle 1Cycle 3Cycle 5Cycle 7 Nr. of Reflns. % of P + > ½ Nr. of Reflns. % of P + > ½ Nr. of Reflns. % of P + > ½ Nr. of Reflns. % of P + > ½ UJZ Phase Statistics

46 residues 13 with side chains MR model MR model Cycle 2 ARP/wARP-DM iteration Cycle 1 Final model Final model 215 residues Cycle 1 Cycle 3 ARP/wARP-OASIS-DM iteration Cycle 7 Cycle residues all with side chains

Hai-fu Fan 1, Yuan-xin Gu 1, Tao Jiang 2, Zheng-jiong Lin 2 & Chao-de Zheng 1 Hai-fu Fan 1, Yuan-xin Gu 1, Tao Jiang 2, Zheng-jiong Lin 2 & Chao-de Zheng 1 Participants of this project Staffs Ph.D. students Jian-rong Chen 1, Qiang Chen 3, Yao He 1, Sheng Huang 1,4, He Li 2, Jia-wei Wang 1, Li-jie Wu 1, De-qiang Yao 1,5 & Tao Zhang 1,6 Jian-rong Chen 1, Qiang Chen 3, Yao He 1, Sheng Huang 1,4, He Li 2, Jia-wei Wang 1, Li-jie Wu 1, De-qiang Yao 1,5 & Tao Zhang 1,6 1 Institute of Physics, CAS, Beijing, China 2 Institute of Biophysics, CAS, Beijing, China 3 Peking University, Beijing, China 4 Institute of High Energy Physics, CAS, Beijing, China 5 Univ. of Science & Technology of China, Hefei, China 6 Lanzhou University, Lanzhou, China 1 Institute of Physics, CAS, Beijing, China 2 Institute of Biophysics, CAS, Beijing, China 3 Peking University, Beijing, China 4 Institute of High Energy Physics, CAS, Beijing, China 5 Univ. of Science & Technology of China, Hefei, China 6 Lanzhou University, Lanzhou, China

SAD data used in this presentation were kindly provided by SAD data used in this presentation were kindly provided by Acknowledgements Dr. Z. Dauter 1, Dr. S. J. Gamblin 2, Dr. B. D. Sha 3, Prof. I. Tanaka 4, Dr. N. Watanabe 4 & Dr. B. Xiao 2 Dr. Z. Dauter 1, Dr. S. J. Gamblin 2, Dr. B. D. Sha 3, Prof. I. Tanaka 4, Dr. N. Watanabe 4 & Dr. B. Xiao 2 1 Argonne National Laboratory, USA 2 The National Institute for Medical Research, UK 3 Department of Cell Biology, University of Alabama at Birmingham, USA 4 Graduate School of Science, Hokkaido University, Japan 1 Argonne National Laboratory, USA 2 The National Institute for Medical Research, UK 3 Department of Cell Biology, University of Alabama at Birmingham, USA 4 Graduate School of Science, Hokkaido University, Japan

Thank you!