Zhang, T., He, Y., Wang, J.W., Wu, L.J., Zheng, C.D., Hao, Q., Gu, Y.X. and Fan, H.F. (2012) Institute of Physics, Chinese Academy of Sciences Beijing,

Zhang, T., He, Y., Wang, J.W., Wu, L.J., Zheng, C.D., Hao, Q., Gu, Y.X. and Fan, H.F. (2012)

Institute of Physics, Chinese Academy of SciencesBeijing, P. R. China

OASIS & IPCAS

OASIS (One-wavelength Anomalous scattering or Single Isomorphous Substitution)

A direct-method phase extension/refinement program based on a partial structure, which can be just the heavy-atom sub-structure or it can be from fragments to a nearly complete model

SAD/SIR information will be beneficial but not compulsory

IPCAS (Iterative Protein Crystal-structure Automatic Solution )

A pipeline from reflection files (*.sca or *.mtz) to a structure model (*.pdb and *.mtz)

Phasing: OASIS, ShelxC/D/E, PHASERDensity modification: DM, RESOLVE

Model building/refinement: ARP/wARP, PHENIX.AutoBuild, Buccaneer

为什么要搞方法研究并发展自动化计算技术？

这是综合竞争能力的

基础之一

结构测定周期

过去现在青霉素～ 3 年 1 ～ 2 天维生素 B12 ～ 8 年 1 ～ 2 天胰岛素～ 14 年～ 1 周肌红 / 血红蛋白

> 20 年～ 1 周

回顾历史

OASIS 的特色直接法相位推演

1. 基于 SAD/SIR 信息 2. 基于部分结构模型或碎片 3. 基于部分已知相位

1947 D. Harker & J. Kasper1952 D. Sayre1950’s J. Karle & H. Hauptman1964 I. L. Karle & J. Karle1970’s M. M. Woolfson1985 Nobel Prize awarded to J. Karle & H. Hauptman

晶体学中的直接法

直接法应用于蛋白质晶体学Ab initio direct methods: SAYTAN (M.M. Woolfson’s team, 1988 ~) SnB (H.A. Hauptman’s team, 1993 ~) SHELXD (G.M. Sheldrick’s team, 1994 ~) ACORN (M.M. Woolfson’s team, 2000 ~)

Direct-method phase extension/refinement:(Combining direct methods with other protein phasing methods) Fan, H.F. Acta Phys. Sin. 21, 1114-1118 (1965) Sayre, D. Acta Cryst. A28, 210-212 (1972) Hauptman, H.A. Acta Cryst. A38, 632-641; 289-294 (1982) Giacovazzo, C. Acta Cryst. A39, 585-592 (1983) Fan, H.F. & Gu, Y.X. Acta Cryst. A41, 280-284 (1985)

OASIS Hao, Q., Gu, Y.X., Zheng, C.D. & Fan, H.F. J. Appl. Cryst. 33, 980-981 (2000)

• SAD/SIR iterationIterative phasing/model building based on the heavy-atom substructure bearing SAD/SIR information

• MR iterationIterative phasing/model building based on a structure model, which can be from fragments to a nearly complete model without SAD/SIR information

• Phase extensionIterative phasing/model building based on a subset of known phases

• SAD/SIR iterationIterative phasing/model building based on the heavy-atom substructure bearing SAD/SIR information

• MR iterationIterative phasing/model building based on a structure model, which can be from fragments to a nearly complete model without SAD/SIR information

• Phase extensionIterative phasing/model building based on a subset of known phases

IPCAS 的特色

‘SAD iteration’ in IPCAS

Acta Cryst. D60, 1991-1996 (2004)Acta Cryst. D62, 883-890 (2006)

OASIS

DMRESOLVE

AutoBuild (Phenix)ARP/wARP/RefMacBuccaneer/RefMac

Direct-method SAD phasing

SAD data

Ok?

End

Partial structure

Density modification

Model buildingand refinement

Yes

No

Bimodal distributionfrom SAD

" " "

The phase ofF”

P

Phase information available in SAD given the known heavy-atom substructure

Phase information available in SAD given the known heavy-atom substructure

Peaked at

"2

jjj

N

jj lzkyhxifiF

ano

2exp"1

"

1

Sim distribution

2 Cochrandistribution

Peaked atany where

from 0 to 2p

3


Acta Cryst. A41, 280-284 (1985)

, 3

1 1tanh sin

2 2

sin sin

best best

P

m m

h h

h' h h' h h' h' h h' hh'

Cochran distribution combined with

bimodal SAD distribution

Sim distribution

Bimodal SAD distribution

" h h h


X-rays l=0.92Å 2.9Å Ba-SAD dataRedundancy=3.9

SCIENCE 306, 104 (2004)

X-rays l=0.9789Å 3.3Å Se-SAD dataRedundancy=3.3

Nat. Struct. Mol. Biol. 13, 589 (2006)

X-rays Cu-Ka2.1Å S-SAD dataActa Cryst. D61, 1533 (2005) Chinese Phys. B 17, 1 (2008)

SAD phasing by OASIS

SAD phasing by OASIS

l=0.9796Å; 2.3ÅSe-SAD dataAuto-Rickshaw builta 57% model beforeand a 89% modelafter using OASIS

J. Mol. Biol. 383, 49–61 (2008)

X-rays Cr-Ka 2.3Å S-SAD data

213 residues

with 2 S atoms in

the ASU 。

Direct method phase

refinement

Ok?

End

Partial structure

Density modification

Model buildingand refinement

‘MR iteration’ in IPCAS

OASIS

DMRESOLVE

AutoBuild (Phenix)ARP/wARP/RefMacBuccaneer/RefMac

Yes

No

, 3

1 1tanh sin

2 2

sin sin best best

P

m m

h h


Partial structure

" h h h

Phasing part of ‘MR iteration’

j”h is a set of ‘reference phases’ calculated each cycle from randomly selected 5% of the current structure model;|Djh| is defined as the absolute difference

between the current phase and j”h

Acta Cryst. D63, 793-799 (2007)

46 residues13 with side chains

MRmodelMRmodel

Cycle 2

ARP/wARP-DMiterationCycle 1

FinalmodelFinalmodel215 residues

Cycle 1 Cycle 3

OASIS-DM-ARP/wARP iteration

Cycle 7Cycle 5201 residuesall with side chains

E7_C–Im7_C E7_C–Im7_C

MR-model completion with E7_C–Im7_C data (1ujz)

Model resulting fromSAD phasing

MR iterationOasis - DM -

Buccaneer - Refmac

Final1086 residues

in the AU

MR iteration based on a Phenix SAD model Tom70p (2gw1) 3.3Å Se-SAD data, redundancy = 3.3

Residues build 1074 906

Residues assigned 1017 207

R / R free0.285 / 0.403 0.48 / 0.59

Model resulting from MRSAD phasing

MR iterationOasis - DM -

Buccaneer - Refmac

Final1356 residues

in the AU

MR iteration based on an Auto-Rickshaw MRSAD model TM1782 (1vkn) 1.8Å Se-SAD data, redundancy = 6.1

Residues build 1369 1234

Residues assigned 1353 1068

R / R free0.230 / 0.300 0.261/0.342

A latest application of MR model completion

MBP-NDM1 Data (2.25Å resolution) was used (Zhang and Hao, The FASEB Journal, 2011)

Target structure Without OASIS With OASIS

, 3

1 1tanh sin

2 2

sin sin

best best

P

m m

h h


As defined in MR iteration

Direct-method phase extension

" h h h

Input known phases to be kept fixed until the resultant model becomes big enough

Initial model

Phenix.Autobuildrunning with ‘quick’

and ‘helixes & strands only’

AutoBuild quick +20 cycles of

Oasis-DM-Buccaneeriteration

Starting with 4.5Å knownphases and2.8Å Fobs

Finalmodel

AutoBuild quick +20 cycles of

DM-Buccaneeriteration

1h3i Phase extension from 4.5 to 2.8Å

An example of direct-method phase extension

SIRAS data

DM, AutoBuild

Partial model

MR completion

Native data

SIRAS phasing SAD phasing

End

Derivative 21

Treatment of SIRAS data

1

DM, AutoBuild

Partial model

MR completion

SIRAS phasing

End

SIRAS data

SAD phasing

DM, AutoBuild

Partial model

MR completion

Native data

End

Derivative 2

SIRAS resultant model improved by the ‘MR iteration’ of OASIS-IPCAS

SIRAS model from2.13Å native &2.65Å Hg-derivative data

Model after ‘MR iteration’based on the SIRAS model and 2.13Å native data

Final model

1

Better results from SAD phasing of the derivative followed by direct-method aided model completion of the native

2

Direct-method SAD phasing of 2.65Å derivative data

Direct-method aided model completion applied to 2.13Å native data

Final model

4.0Å model from direct-method SAD phasing

2.13Å model after direct-method phase extension

2.13Å model after direct-method aided model completion

SIRAS data with derivative at 4Å resolution– a simulation: Hg-derivative data truncated to 4.0Å

Final Model

谢谢！谢谢！

Documents

Zhang, T., He, Y., Wang, J.W., Wu, L.J., Zheng, C.D., Hao, Q., Gu, Y.X. and Fan, H.F. (2012) Institute of Physics, Chinese Academy of Sciences Beijing,