SAD phasing by OASIS-2004 SAD phasing by OASIS-2004

SAD phasingby OASIS-2004

SAD phasingby OASIS-2004

OASIS-2004OASIS-2004

Freely available at http://cryst.iphy.ac.cn

A program for direct-method phasing and reciprocal-space fragment extension with

SAD/SIR data

By J.W. Wang, Y.X. Gu*, C.D. Zheng, H.F. Fan*and Q. Hao

* Corresponding authors

SAD data used in this presentationwere kindly provided by

Dr. Z. Dauter, Dr. S. J. Gamblin, Prof. S. Hasnain, Prof. I. Tanaka, Dr. N. Watanabe, Dr. M.S. Weiss, D

r. B. Xiao and Dr. C. Yang

Calculations were done by

Mr. D.Q. Yao, Dr. S. Huang and Dr. J.W. Wang

Acknowledgements

Two examples ondifficult SAD phasing

Two examples ondifficult SAD phasing

OASIS-2004application

Contoured at 1

XylanaseSpace group: P21 Unit cell: a = 41.07, b = 67.14, c = 50.81Å = 113.5o Resolution limit: 1.75Å; Multiplicity: 15.9Anomalous scatterer: S (5 ) X-rays:synchrotron radiation= 1.488Å; f ” = 0.52Bijvoet ratio: <|F |>/<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

Data courtesy of Professor Isao Tanaka & Dr. Nobuhisa WatanabeGraduate School of Science, Hokkaido University, Japan

Space group: P21212 Unit cell: a = 100.2639 b = 108.9852 c = 114.6272ÅNumber of residues in the ASU: 1206Resolution range: 50.00-2.01ÅMultiplicity: 20.9Anomalous scatterer: S (22) Wavelength: = 2.291Å; f ” = 1.14Bijvoet ratio: <|F|>/<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-2004application

Features ofFeatures of

OASIS-2004OASIS-2004

1. Better initial SAD phases1. Better initial SAD phases

" h h h" h h h

1tan( ) 2 ( ) sin cos

2best P

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

12 21 1

exp 2 2 1 cos2 cos22 2

m P

2h h h h h

, 3

1 1tanh sin

2 2

sin sinbest bes

c

t

P

m m

h h

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

, 3

1 1tanh sin

2 2

sin sinbest bes

c

t

P

m m

h h

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

Bimodal distributionof SAD

" " "

The phase ofF”

Phase information available in SAD

Cochrandistribution

Peaked atany where

from 0 to 2

Peaked at

"2

Sim distribution

Two different kinds of initial SAD phases

P++P

PSim PBimodal Sim-modified phases

P+-modified phases

P+

PSim PCochran

Se-SAD

Histone Methyltransferase Set 7/9Space group: P212121

Unit cell: a = 66.09, b = 82.83, c = 116.15ÅNumber of residues in ASU: 560

Number of independent reflections: 16352Resolution limit: 2.8Å

Multiplicity: 3.8Anomalous scatterer: Se(12)

= 0.9794Å; f’ = -7.5, f” = 6.5 Bijvoet ratio: <|F|>/<F> = 7.03%

SAD phasing by OASIS-2004 + DMData provided by Dr. S. J. Gamblin

and Dr. B. Xiao

Comparison of the two kinds of initial phasesusing 4 typical reflections from the protein

histone methyltransferase SET 7/9

Comparison on cumulative phase errors sorted in descending order of Fobs

Comparison on cumulative phase errors sorted 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 ))

2

22

2 "

Fn

F

h

h

h

2

22

2 "

Fn

F

h

h

h

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

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

1 2

21 1

exp 2 2 1 cos2 cos22 2

m P

2h h h h

1 22

1 1exp 2 2 1 cos2 cos2

2 2m P

2h h h h

, 3

1 1

2 2

tanh sin sin sinbest best

P

m m

h

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

, 3

1 1

2 2

tanh sin sin sinbest best

P

m m

h

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

1exp 2

2 2h 1

exp 22

2h

Automatic solution of protein structures

OASIS-2004 DM

Automatic solution of protein structures

OASIS-2004 DM

by a single run ofby a single run of

RESOLVE(Build only)

and/or

ARP/wARP

RESOLVE(Build only)

and/or

ARP/wARP

++ ++

Br-SADOASIS-2004application

Contoured at 1

Pepstatin-insenstive carboxylproteinaseSpace group: P62

Unit cell: a = b = 97.31, c = 82.94Å, = 120o

Resolution limit: 1.8Å; Multiplicity: 5.45Anomalous scatterer: Br (13)X-rays:synchrotron radiation= 0.9191Å; f ” = 5.0 Bijvoet ratio: <| F |>/<F > = 7.06% Phasing: OASIS-2004 + DM (Cowtan)Model building: ARP/wARP found 358 of the total 372 residuesData courtesy of Dr. Z. Dauter, National Cancer Institute, USA

Xe-SADOASIS-2004application

Contoured at 1

Porcine Pancreatic Elastase Space group: P212121 Unit cell: a = 50.2, b = 58.1, c = 74.3ÅResolution limit: 1.94Å; Total rotation range: 360o

Anomalous scatterer: Xe (1)X-rays:synchrotron radiation= 2.1Å; f ” = 11.8 Bijvoet ratio: <| F |>/<F > = 5.76% Phasing: OASIS-2004 + DM (Cowtan)Model building: ARP/wARP found 236 of the total 240 residuesData courtesy of Dr. M. S. Weiss, EMBL Hamburg Outstation, c/o DESY, Germany

S-SADOASIS-2004application

Contoured at 1

Lysozyme Space group: P43212

Unit cell: a = 78.81, c = 36.80ÅAtoms in the asymmetric unit: 1001Resolution limit: 1.53Å; Multiplicity: 23Anomalous scatterer: S (10), Cl (7)X-rays:synchrotron radiation= 1.54Å; f ” = 0.56, 0.70 Bijvoet ratio: <| F |>/<F > = 1.55% Phasing: OASIS-2004 + DM (Cowtan)Model building: ARP/wARP found 128 of the total 129 residuesData courtesy of Dr. Z. Dauter, National Cancer Institute, USA

Cu-K

Fe-SAD

302 residues found automatically

YfbpASpace group: P212121 Unit cell: a = 42.792, b = 54.134, c = 115.222ÅResolution range: 57.74 – 1.42ÅAnomalous scatterer: Fe (1)Wavelength: 1.542Åf ” = 3.20<|F|>/<F> ~ 1.4%Phased by: OASIS + DM (Cowtan)Automatic model building by: ARP/wARPData provided by Dr. Cheng Yang Rigaku/MSC, USA

OASIS-2004 application

3. Dual-space fragment extension3. Dual-space fragment extension

, 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'

, 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'

PartialmodelPartialmodel

PartialmodelPartialmodel

NoNoYesYes

Reciprocal-spacefragment extension by

OASIS-2004 + DM

Reciprocal-spacefragment extension by

OASIS-2004 + DM

Real-space fragment extension by

RESOLVE BUILD and/or ARP/wARP

Real-space fragment extension by

RESOLVE BUILD and/or ARP/wARP

OK?OK?

End End

Glucoseisomerase

S-SADCu-K

17%Cycle 097%Cycle 6

Glucoseisomerase

S-SADCu-K

Cr-K Se, S-SAD Alanine racemase

Cycle 052%

Cr-K Se, S-SAD Alanine racemase

Cycle 497%

25%Cycle 0

Xylanase S-SADSynchrotron = 1.49Å

Xylanase S-SADSynchrotron = 1.49Å

99%Cycle 6

52%Cycle 0

LysozymeS-SADCr-K

LysozymeS-SADCr-K

98%Cycle 6 Azurin

Cu-SADSynchrotron = 0.97Å

Cycle 042%

AzurinCu-SADSynchrotron = 0.97Å

Cycle 395%

Case studyCase study1. Azurin

2. Xylanase1. Azurin

2. Xylanase

Contoured at 1

Space group: P4122 Unit cell: a = b = 52.65, c = 100.63ÅResolution limit: 1.9Å; Multiplicity: 10.0 Anomalous scatterer: Cu (1)X-rays: Synchrotron radiation =0.97Å;f” = 2.206; <|F|>/<F> = 1.44%Phasing: OASIS-2004 + DM (Cowtan)Model building: RESOLVE BUILD and ARP/wARP116 of 129 residues found automaticallyData courtesy of Professor S. Hasnain

Azurin

70

60

50

40

30

80

OASIS-2004

DM

Partial model from RESOLVE BUILD or ARP/wARP

Cycle0 1 2 43

Ph

ase

erro

r in

deg

rees

OASIS-DM-(RESOLVE BUILD, ARP/wARP) IterationAzurin copper-SAD phasing

Synchrotron radiation = 0.97Å, <F>/<F> = 1.44%P

has

e er

ror

in d

egre

es

What would it bewithout using

RESOLVE (build only)?

Is OASISnecessaryhere? Is OASIS

necessaryhere?

OASIS-DM-ARP/wARP IterationAzurin copper-SAD phasing

Synchrotron radiation = 0.97Å, <F>/<F> = 1.44%

OASIS-DM-ARP/wARP IterationAzurin copper-SAD phasing

Synchrotron radiation = 0.97Å, <F>/<F> = 1.44%P

has

e er

ror

in d

egre

es

0 102 4 86

Cycle

70

60

50

40

30

80

70

60

50

40

30

80

OASIS-2004

DM

Partial model from ARP/wARP

Cycle 0(ARP/wARP)

Cycle 4(ARP/wARP)

Cycle 6(ARP/wARP)

Cycle 8(ARP/wARP)

OASIS-DM-ARP/wARP Iteration

Cycle 0(RESOLVE BUILD)

Cycle 1(ARP/wARP)

Cycle 2(ARP/wARP)

Cycle 3(ARP/wARP)

OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration

Cycle 10(ARP/wARP)

Cycle 4(ARP/wARP)

Cycle 0Cycle 0 Cycle 2Cycle 2 Cycle 4Cycle 4

Improvement on electron-density map andautomatic model building

Improvement on electron-density map andautomatic model building

Cycle 0Cycle 0 Cycle 2Cycle 2 Cycle 4Cycle 4

Xylanase

Contoured at 1

Space group: P21 Unit cell: a = 41.07, b = 67.14, c = 50.81Å = 113.5o Resolution limit: 1.75Å; Multiplicity: 15.9Anomalous scatterer: S (5 ) X-rays:synchrotron radiation= 1.488Å; f ” = 0.52Bijvoet ratio: <|F |>/<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

OASIS-2004

DM

Partial model from RESOLVE BUILD or ARP/wARP

Ph

ase

erro

r in

deg

rees

70

60

50

40

30

20

80

10

Cycle0 51 62 43

OASIS-DM-(RESOLVE BUILD, ARP/wARP) IterationXylanase sulfur-SAD phasing

Synchrotron radiation = 1.49Å, <F>/<F> = 0.56%Is OASISnecessaryhere?

What would it bewithout using

RESOLVE (build only)?

Is OASISnecessaryhere?

OASIS-DM-ARP/wARP IterationXylanase sulfur-SAD phasing

Synchrotron radiation = 1.49Å, <F>/<F> = 0.56%

OASIS-DM-ARP/wARP IterationXylanase sulfur-SAD phasing

Synchrotron radiation = 1.49Å, <F>/<F> = 0.56%

70

60

50

40

30

20

80

0 102 124 86 14 16

Ph

ase

erro

r in

deg

rees

Cycle

OASIS-2004

DM

Partial model from ARP/wARP

Cycle 14(ARP/wARP)

Cycle 16(ARP/wARP)

Cycle 17(ARP/wARP)

Cycle 0(ARP/wARP)

Cycle 0(RESOLVE BUILD)

Cycle 3(ARP/wARP)

Cycle 5(ARP/wARP)

Cycle 6(ARP/wARP)

OASIS-DM-ARP/wARP Iteration

OASIS-DM-(RESOLVE BUILD, ARP/wARP) Iteration

Cycle 0Cycle 0 Cycle 3Cycle 3 Cycle 6Cycle 6

Improvement on electron-density map andautomatic model building

Improvement on electron-density map andautomatic model building

ConclusionConclusion

1. OASIS is essential for initial SAD phasing and for initiating a successful dual-space fragment

extension;

2. Starting models from RESOLVE (build only) lead much faster to the final solution;

3. ARP/wARP models with dummy atoms only may lead to nearly the complete structure after

sufficient cycles of iteration.

Thank you!Thank you!