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, 2006Yokohama, Japan

October 19-20, 2006

OASIS-2006OASIS-2006Institute of Physics

Chinese Academy of SciencesBeijing 100080, P.R. China

Institute of PhysicsChinese Academy of Sciences

Beijing 100080, P.R. China

http://cryst.iphy.ac.cnhttp://cryst.iphy.ac.cn

OASIS (2000)OASIS (2000) in CCP4in CCP4

OASIS-2004OASIS-2004 on the Webon the Web

OASIS-2006OASIS-2006 on the Web soonon the Web soon

OASIS-2006 GUI for CCP4iOASIS-2006 GUI for CCP4i

1. Direct-method

2. Reciprocal-space

1. Direct-method

2. Reciprocal-space

Functions of OASIS Functions of OASIS

SAD/SIR PhasingSAD/SIR Phasing

Fragment ExtensionFragment ExtensionFragment ExtensionFragment ExtensionDual-spaceDual-space

by combining with

DM, RESOLVE and ARP/wARPby combining with

DM, RESOLVE and ARP/wARP

Why direct methods?Why direct methods?

For better initial SAD phases!

For better initial SAD phases!

Bimodal distributionfrom SAD

" " "

The phase ofF”

P

Phase information available in SADPhase information available in SAD

Cochrandistribution

Peaked atany where

from 0 to 2

Peaked at

"2

Sim distribution

Two different kinds of initial SAD phasesTwo different kinds of initial SAD phases

P+-modified phases

P++P

PSim PBimodal Sim-modified phases

P+

PSim PCochran

" h h h1

" is the phase of " "exp 2N

j jj

i f i

h h h rF

1 1cos 2 " cos2 "

F h

h h h hh

F F FF

2 ' 'E E E h h h h 3 ' '" " " h h h h

1tan( ) 2 ( ) sin cos

2best P

h h h h

1

2 21 1exp 2 2 1 cos2 cos2

2 2m P

2h h h h h

,

2 2

2U T

pN N

j jj j

E E

Z Z

h h ' " h h h

, 3

1 1tanh sin

2 2

sin sin

c

best best

P

m m

h h

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

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

Comparison of cumulative phase errors in descending order of Fobs

Comparison of cumulative phase errors in descending order of Fobs

60.263.415000

58.461.913500

56.960.812000

62.365.216352

55.659.410500

54.158.79000

52.957. 87500

51.257.06000

50.056.54500

49.157.13000

45.857.11500

Errors of PErrors of P++-modified -modified

phases phases (( oo ))Number of reflectionsNumber of reflections Errors of Sim-modified Errors of Sim-modified

phases phases (( oo ))

Histone methyltransferase SET 7/9

Why dual-space?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 extensionDual-space fragment extension

, 3

1 1tanh sin

2 2

s s inin

best best

P

m m

h h

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

, 3

1 1tanh sin

2 2

s s inin

best best

P

m m

h h

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

Real-spacefragment extension

RESOLVE BUILD and/or ARP/wARP

Real-spacefragment extension

RESOLVE BUILD and/or ARP/wARP

Partialstructure

Partialstructure

NoNo

YesYes

OK?OK?

EndEnd

PartialmodelPartialmodel

OASIS ApplicationsOASIS Applications

Ab initio SAD phasingAb initio SAD phasingwith

weak anomalous signalwith

weak anomalous signal

SHARP-SOLOMON-ARP/wARPSHARP-SOLOMON-ARP/wARP

XylanaseSpace group: P21 Unit cell: a = 41.07, b = 67.14, c = 50.81Å = 113.5o Number of residues in the ASU: 303 Resolution limit: 1.75Å; Multiplicity: 15.9Anomalous scatterer: S (5 ) X-rays:synchrotron radiation= 1.488Å; f ” = 0.52Bijvoet ratio: <|F |>/<F > = 0.56% Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA

OASIS-DM-RESOLVE BUILDcycle 025%

OASIS-DM-ARP/wARPcycle 699%SHARP-DM-RESOLVE BUILDBP3-DM-RESOLVE BUILD

2.1Å 3.5Å 4.0Å

SAD phasing at different resolutionsTT0570 Cu-Kdata, <|F|>/<F> ~ 0.55%

SAD phasing at different resolutionsTT0570 Cu-Kdata, <|F|>/<F> ~ 0.55%

OASIS + DM OASIS + DM

SOLVE/RESOLVE resultsSOLVE/RESOLVE results

improved byimproved by

SOLVE/RESOLVE

SOLVE/RESOLVE +OASIS-DM-RESOLVE (BUILD)

Dual-space fragment extension based on SOLVE/RESOLVE resultsDual-space fragment extension based on SOLVE/RESOLVE results1LIA (d14) 2.8Å SIR data

SOLVE/RESOLVE map

Sigma_A map based on a model manually builtfrom the SOLVE/RESOLVE map

OASIS-DM mapbased on

the same model

Dual-space fragment extension based on SOLVE/RESOLVE resultsDual-space fragment extension based on SOLVE/RESOLVE results2GW1 3.3Å SAD data

Dual-space fragment extension based on SOLVE/RESOLVE resultsDual-space fragment extension based on SOLVE/RESOLVE results2GW1 3.3Å SAD data

Sigma_A map based on a model manually builtfrom the SOLVE/RESOLVE map

SOLVE/RESOLVE map OASIS-DM mapbased on

the same model

Molecular Replacement Molecular Replacement

Fragment extensionFragment extension

based onbased on

Fragment extension based on

molecular replacement

Finalmodel

acidic phospholipase A2124 residues

MRmodel

60 residues48 residues

ARP/wARP-OASIS-DM iteration

Cycle 1 Cycle 2 Cycle 3

ARP/wARP-DM iteration

Cycle 9 Cycle 11 Cycle 13

ARP/wARP-DM iteration

A difficult case: 1UJZA difficult case: 1UJZSpace group: I 222Unit cell: a=62.88, b=74.55, c=120.44ÅNumber of residues in ASU: 215 molecule A: 87 residues molecule B: 128 residuesNumber of residues in the starting model: 46 (all in Mol. A)Resolution range: 37.57 – 2.10ÅNumber of reflections: 16460

Space group: I 222Unit cell: a=62.88, b=74.55, c=120.44ÅNumber of residues in ASU: 215 molecule A: 87 residues molecule B: 128 residuesNumber of residues in the starting model: 46 (all in Mol. A)Resolution range: 37.57 – 2.10ÅNumber of reflections: 16460

Fragment ExtensionFragment ExtensionDual-spaceDual-space

without SAD/SIR informationwithout SAD/SIR information

, 3

1 1tanh sin

2 2

sin si n

best best

P

m m

h h

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

Partialstructure

Partialstructure

" h 5%

the phase of atoms

randomly selected from the current model

" h h h

" . . "model modeli e h h h h

Density modification

by DM

Density modification

by DM

NoNo

MRmodel

MRmodel

YesYes

EndEnd

Model rebuild by

RESOLVE BUILD or ARP/wARP

Model rebuild by

RESOLVE BUILD or ARP/wARP

OK?OK?

Phase improvement

by OASIS

Phase improvement

by OASIS

P+ > 0.5

” model

P+ < 0.5

” model

<||> ~

<||> ~

”

Range of phase error in degrees

Cycle 1 Cycle 3 Cycle 5 Cycle 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

0 - 30 3337 55 3761 60 4197 65 5733 76

30 - 60 2856 48 2874 49 3049 52 2469 43

60 - 90 2635 38 2359 38 2279 35 1852 18

90 - 120 2281 31 2189 27 1975 24 1702 7

120 -150 2175 24 2097 21 1858 16 1813 6

150 - 180 2064 22 2071 17 1991 13 1779 5

1UJZ Phase Statistics1UJZ Phase Statistics

46 residues13 with side chains

MRmodelMRmodel

Cycle 2

ARP/wARP-DMiterationCycle 1

FinalmodelFinalmodel

215 residues

Cycle 1 Cycle 3

ARP/wARP-OASIS-DM iteration

Cycle 7Cycle 5201 residuesall with side chains

Hai-fu Fan1, Yuan-xin Gu1, Tao Jiang2, Zheng-jiong Lin2 & Chao-de Zheng1

Hai-fu Fan1, Yuan-xin Gu1, Tao Jiang2, Zheng-jiong Lin2 & Chao-de Zheng1

Participants of this projectParticipants of this projectStaffsStaffs

Ph.D. studentsPh.D. studentsJian-rong Chen1, Qiang Chen3, Yao He1,

Sheng Huang1,4, He Li2, Jia-wei Wang1, Li-jie Wu1, De-qiang Yao1,5 & Tao Zhang1,6

Jian-rong Chen1, Qiang Chen3, Yao He1, Sheng Huang1,4, He Li2, Jia-wei Wang1, Li-jie Wu1,

De-qiang Yao1,5 & Tao Zhang1,6

1 Institute of Physics, CAS, Beijing, China2 Institute of Biophysics, CAS, Beijing, China3 Peking University, Beijing, China4 Institute of High Energy Physics, CAS, Beijing, China5 Univ. of Science & Technology of China, Hefei, China6 Lanzhou University, Lanzhou, China

1 Institute of Physics, CAS, Beijing, China2 Institute of Biophysics, CAS, Beijing, China3 Peking University, Beijing, China4 Institute of High Energy Physics, CAS, Beijing, China5 Univ. of Science & Technology of China, Hefei, China6 Lanzhou University, Lanzhou, China

SAD data used in this presentationwere kindly provided by

SAD data used in this presentationwere kindly provided by

AcknowledgementsAcknowledgements

Dr. Z. Dauter1, Dr. S. J. Gamblin2, Dr. B. D. Sha3, Prof. I. Tanaka4, Dr. N. Watanabe4 & Dr. B. Xiao2

Dr. Z. Dauter1, Dr. S. J. Gamblin2, Dr. B. D. Sha3, Prof. I. Tanaka4, Dr. N. Watanabe4 & Dr. B. Xiao2

1 Argonne National Laboratory, USA2 The National Institute for Medical Research, UK3 Department of Cell Biology, University of Alabama at Birmingham, USA4 Graduate School of Science, Hokkaido University, Japan

1 Argonne National Laboratory, USA2 The National Institute for Medical Research, UK3 Department of Cell Biology, University of Alabama at Birmingham, USA4 Graduate School of Science, Hokkaido University, Japan

Thank you!Thank you!