OASIS What is it? How it works? What next? What is it? How it works? What next?

Two categories of Direct Methods in Protein Crystallography Ab initio direct methods: SnB (H.A. Hauptman’s team) ShelxD (G.M. Sheldrick’s team) Acorn (M.M. Woolfson’s team) OASIS A program of direct-method phasing A program of direct-method phasing based on a partial model with or without SAD/SIR information

Features Phase derivation based on a partial model with or without SAD/SIR information breaking the SAD/SIR phase ambiguity phase flipping of the partial model SAD/SIR iteration iterative SAD/SIR phasing and model building MR iteration iterative phasing and model building without using SAD/SIR information Phase derivation based on a partial model with or without SAD/SIR information breaking the SAD/SIR phase ambiguity phase flipping of the partial model SAD/SIR iteration iterative SAD/SIR phasing and model building MR iteration iterative phasing and model building without using SAD/SIR information

OASIS What it is? How it works? What next? What it is? How it works? What next?

Breaking the SAD phase ambiguity TT0570 (2d5w) sulfur-SAD CuK  data, redundancy = 26.1 (1206 resides in the AU, Bijvoet ratio = 0.55%) Programs used: Oasis, DM, FFT program in CCP4 and PyMOL Chinese Physics B 17, 1-9 (2008)

Iterative SAD phasing & model building TT0570 (2d5w) sulfur-SAD CuK  data Programs used: Oasis, DM, Resolve (build only), RefMac and ARP/wARP or Buccaneer Final 2.2 Å ARP/wARP 2.0 Å Buccaneer 2.5 Å Buccaneer 3.0 Å

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

Dual-space SAD/SIR iteration Acta Cryst. D60, (2004) Acta Cryst. D62, (2006)

Reciprocal space ： Replacing |F c | with |F o | and modifying  Real space ： modifying   (r) |F o |exp(i  )   (r) |F c |exp(i  )    (r) Dual-space iteration  a phasing framework

Dual-space iterative phasing Gerchberg, R.W. & Saxton, W.O. Optik, 34, (1971) ； 35, (1972) Wang, B.C. Methods in Enzymology 115, (1985) Weeks, C. M., Detita, G. T., Miller, R. & Hauptman, H. A. Acta Cryst. D49, (1993) Sheldrick, G. M., Hauptman, H. A., Weeks, C. M., Miller, R. & Usoâ N, I. (2001). International Tables for Crystallography Vol. F, edited by E. Arnold & M. Rossmann, pp Wang, J.W., Chen, J. R., Gu, Y. X., Zheng, C. D. & Fan, H. F. Acta Cryst. D60, 1991–1996 (2004) SAD/SIR phasing + model completion (fragment extension)

Dual-space MR iteration Acta Cryst. D63, (2007)

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 OASIS - DM-ARP/wARP iteration Cycle 7 Cycle residues all with side chains E7_C–Im7_C MR-model completion with E7_C–Im7_C data (1ujz)

Flipping 范海福 《物理学报》 21, (1965) (in Chinese) Hai-fu Fan, Chinese Phys (1965) Sign flipping Weeks, C. M., Detita, G. T., Miller, R. & Hauptman, H. A. Acta Cryst. D49, (1993) Phase flipping Sheldrick, G. M., Hauptman, H. A., Weeks, C. M., Miller, R. & Usoâ N, I. (2001). International Tables for Crystallography Vol. F, edited by E. Arnold & M. Rossmann, pp Atom flipping Oszlányi, G. & Sütő, A. Acta Cryst. A60, (2004) Charge flipping He, Y., Yao, D.Q., Gu, Y. X., Lin, Z.J., Zheng, C. D. & Fan, H. F. Acta Cryst. D63, (2007) Phase flipping

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

Percentage of residues assigned Cycle Tom70p 3.3Å Se-SAD data X-Ray wavelength: Å Redundancy: 3.3 Number of residues in the AU: Cycles SAD iteration 9 Cycles MR iteration based on 7 cycles of SAD iteration SAD iteration: Oasis - DM - Resolve (build only) - Buccaneer - Refmac Heavy-atom sites: located by 100 trials of ShelxD and refined by Solve MR iteration: Oasis - DM - Buccaneer - Refmac Combining SAD iteration and MR iteration Chin. Phys. B 19, (2010)

SAD iteration Oasis - DM - Resolve (build only) - Buccaneer - Refmac Final 1086 residues in the AU MR iteration based on SAD iteration Oasis - DM - Buccaneer - Refmac MR iteration based on Oasis SAD phasing Tom70p 3.3Å Se-SAD data (2gw1) Residues build Residues assigned R / R free / / 0.435

Examples on model completion by the program combination of OASIS (phasing) DM (density modification) Buccaneer (model building) RefMac (refinement) with starting models from different program packages without involving OASIS

Model resulting from SAD phasing MR iteration Oasis - DM - Buccaneer - Refmac Final 1086 residues in the AU MR iteration based on a SAD model Tom70p (2gw1) 3.3Å Se-SAD data, redundancy = 3.3 Residues build Residues assigned R / R free / / 0.59

MR-iteration based on a MAD model Set9/7 (1h3i) 2.8Å data Model resulting from MAD phasing MAD model extended after 7 cycles iteration of Oasis - DM - Buccaneer - Refmac Final 586 residues in the AU Residues build Residues assigned R / R free / / 0.374

Residues build Residues assigned R / R free / / Model resulting from MIR phasing MIR model extended after 9 cycles of Oasis - DM - Buccaneer - Refmac Final 668 residues in the AU MR-iteration based on an MIR model Rpe (1lia) 2.8Å data

Model resulting from MRSAD phasing MR iteration Oasis - DM - Buccaneer - Refmac Final 1356 residues in the AU MR iteration based on an MRSAD model TM1782 (1vkn) 1.8Å Se-SAD data, redundancy = 6.1 Residues build Residues assigned R / R free / /0.342

MR iteration based on an MRSAD model TM0119 (2f4l) 2.5Å Se-SAD data, redundancy = 3.8 Residues build Residues assigned R / R free / /0.300 MR iteration Oasis - DM - Buccaneer - Refmac Final 1140 residues in the AU Model resulting from MRSAD phasing

Further improve the phasing algorithm Allow using a rough electron density map instead of a partial model during the iteration Combine oversampling procedure with direct-method phasing Further improve the phasing algorithm Allow using a rough electron density map instead of a partial model during the iteration Combine oversampling procedure with direct-method phasing What next?

University of Hong Kong, China Acknowledgements Professor Quan Hao Institute of Physics, Chinese Academy of Sciences, Beijing, China Professor Yuan-xin Gu, Dr. Jia-wei Wang, Dr. Yao He, Dr. Tao Zhang The project is supported by the Chinese Academy of Sciences and the 973 Project (Grant No. 2002CB713801) of the Ministry of Science and Technology of China. Institute of Biophysics, Chinese Academy of Sciences, Beijing China Professor Zheng-jiong Lin, Dr. Qiu-ying Yang EMBL-Hamburg Outstation, Hamburg, Germany Dr. Santosh Panjikar

Thank you!