In honor of Professor B.C. Wangreceiving the 2008 Patterson AwardIn honor of Professor B.C. Wang

receiving the 2008 Patterson Award

Direct Methods andSolvent Flattening

Direct Methods andSolvent Flattening

Q. Hao, Y.X. Gu, C.D. Zheng & H.F. FanQ. Hao, Y.X. Gu, C.D. Zheng & H.F. Fan

the SIR/SAD phase ambiguity the SIR/SAD phase ambiguity

Direct methods breakingDirect methods breaking

' ' ' ' ,' '

' ' ,'

1

21

unknown unknownknown

known

unknown unknownknown

known

A A A B B AV

B A B BV

h h h h h h h hh h

h h h h hh

Component Relationships (1965)Component Relationships (1965)

Chinese Physics 1429-1435 (1965)

SIR case:

If the heavy-atom substructure is centrosymmetric,

we know Bh and |Ah|, the question is to find the sign of Ah .

SAD case:

If the heavy-atom substructure is centrosymmetric,

we know Ah and |Bh|, the question is to find the sign of Bh .

P+ formula (1985)P+ formula (1985)

Acta Cryst. A40, 489-495 (1984)Acta Cryst. A40, 495-498 (1984)Acta Cryst. A41, 280-284 (1985)

' ' , ' 3 ', ','

1 1tanh sin

2 2

sin ' sinbest best

P

m m

h h

h h h h h h h h hh

' h h h The phase problem reduces to a sign problem

A combination of SIR/SAD bimodal distribution with theCochran distribution incorporating with partial structure information

Direct-method phasing of the 2Å experimental SAD data of the protein aPP

Direct-method phasing of the 2Å experimental SAD data of the protein aPP

Avian Pancreatic Polypeptide

Space group: C2 Unit cell: a = 34.18, b = 32.92, c = 28.44Å; = 105.3o

Protein atoms in ASU: 301Resolution limit: 2.0ÅAnomalous scatterer: Hg (in centric arrangement)Wavelength: 1.542Å (Cu-K) f” = 7.686Locating heavy atoms & SAD phasing: direct methods

Acta Cryst. (1990). A46, 935.

Avian Pancreatic Polypeptide

Space group: C2 Unit cell: a = 34.18, b = 32.92, c = 28.44Å; = 105.3o

Protein atoms in ASU: 301Resolution limit: 2.0ÅAnomalous scatterer: Hg (in centric arrangement)Wavelength: 1.542Å (Cu-K) f” = 7.686Locating heavy atoms & SAD phasing: direct methods

Acta Cryst. (1990). A46, 935. Data courtesy of Professor Tom BlundellData courtesy of Professor Tom Blundell

OASIS + DM (Cowtan, CCP4)OASIS + DM (Cowtan, CCP4)

SAD phasing bySAD phasing by

Much more complicated proteinshave been solved by combining

direct methods & solvent flattening:

Much more complicated proteinshave been solved by combining

direct methods & solvent flattening:

OASIS-2006OASIS-2006Institute of Physics

Chinese Academy of SciencesBeijing 100080, P.R. China

Institute of PhysicsChinese Academy of SciencesBeijing 100080, P.R. China

http://cryst.iphy.ac.cnhttp://www.ccp4.ac.uk/prerelease

http://cryst.iphy.ac.cnhttp://www.ccp4.ac.uk/prerelease

TTHA1634 fromThermus thermophilus HB8

Data courtesy of Professor Nobuhisa WatanabeDepartment of Biotechnology and Biomaterial Chemistry, Nagoya University, Japan

Space group: P21212 Unit cell: a = 100.57, b = 109.10, c = 114.86ÅNumber of residues in the ASU: 1206Resolution limit: 2.1ÅMultiplicity: 29.2Anomalous scatterer: S (22) X-ray wavelength: = 1.542Å (Cu-K)Bijvoet ratio: <|F|>/<F> = 0.55%Phasing method: A single run of OASIS2006 + DM (Cowtan)Model building: ARP/wARP

ARP/wARP found 1178 of the total 1206 residues,all docked into the sequence.

Ribbon model plotted by PyMOL

What’s thelow resolution limit ?

What’s thelow resolution limit ?

SAD phasing at different resolutionsTTHA1634 Cu-Kdata, <|F|>/<F> ~ 0.55%

SAD phasing at different resolutionsTTHA1634 Cu-Kdata, <|F|>/<F> ~ 0.55%

2.1Å

3.0Å

3.5Å

4.0Å

Very good

Good

Marginally traceable

Still informative

Maps at 1 phased by a single run of OASIS + DM (Cowtan) plotted by PyMOL

OASIS, DM, RESOLVE & ARP/wARPOASIS, DM, RESOLVE & ARP/wARP

SAD phasing + Dual-space fragment extension by combination of

SAD phasing + Dual-space fragment extension by combination of

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

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%

Ribbon models plotted by PyMOL

Data courtesy of Professor N. Watanabe,Professor S. Hasnain, Dr. Z. Dauter andDr. C. Yang

Direct-method aided

MR-model completion

Direct-method aided

MR-model completion

Dual-space fragment extension

without SAD/SIR information

Dual-space fragment extension

without SAD/SIR information

46 residues13 with side chains

MRmodelMRmodel

Cycle 2

ARP/wARP-DMiterationCycle 1

Cycle 1 Cycle 3

ARP/wARP-OASIS-DM iteration

Cycle 7Cycle 5201 residuesall with side chains Final

modelFinalmodel

215 residues

1UJZ

Ribbon models plotted by PyMOL

dealing with low resolution SIR/SAD data

dealing with low resolution SIR/SAD data

Combining SOLVE/RESOLVE and OASIS + DM

Combining SOLVE/RESOLVE and OASIS + DM

R-phycoerythrinSIR data from the native and thep-chloromercuriphenyl sulphonic acid derivative

Space group: R3Unit cell: a = b = 189.8, c = 60.0Å;     = 120o

Number of residues in the ASU: 668 Resolution limit: 2.8ÅReplacing atoms: HgX-rays: Cu-K, λ = 1.542Å

J.Mol.Biol. 262 721-731 (1996)Chinese Physics 16, 3022-3028 (2007)

SOLVE/RESOLVE SOLVE/RESOLVE& OASIS + DM

Maps plotted by PyMOL

SOLVE/RESOLVE SOLVE/RESOLVE& OASIS + DM SOLVE/RESOLVE

SOLVE/RESOLVE &OASIS + DM

Tom70pSpace group: P21

Unit cell: a = 44.89, b = 168.8, c = 83.4Å;    β = 102.74o

Number of residues: 1086 Resolution limit: 3.3ÅMultiplicity: 3.3Anomalous scatterer: Se (24)X-rays: Synchrotron, λ = 0.9789Å, Δf" = 6.5Bijvoet ratio: <|ΔF|>/<|F|> = 4.3%

Nature Structural & Molecular Biology 13, 589-593 (2006)Chinese Physics B 17, 1-9 (2008)

Maps plotted by PyMOL

Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

AcknowledgementsAcknowledgementsProfessor Zhengjiong LinProfessor Zhengjiong Lin

1 Beijing National Laboratory for Condensed Matter Physics,

Institute of Physics, Chinese Academy of Sciences, China2 National Laboratory of Protein Engineering and Plant Genetic

Engineering, Peking University, Beijing, China3 Institute of Biophysics, Chinese Academy of Sciences, Beijing China

1 Beijing National Laboratory for Condensed Matter Physics,

Institute of Physics, Chinese Academy of Sciences, China2 National Laboratory of Protein Engineering and Plant Genetic

Engineering, Peking University, Beijing, China3 Institute of Biophysics, Chinese Academy of Sciences, Beijing China

Drs Y. He1, D.Q. Yao1, J.W. Wang1, S. Huang1, J.R. Chen1, Q. Chen2, H. Li3, Prof. T. Jiang3, & Mr. T. Zhang1

Drs Y. He1, D.Q. Yao1, J.W. Wang1, S. Huang1, J.R. Chen1, Q. Chen2, H. Li3, Prof. T. Jiang3, & Mr. T. Zhang1

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.

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.

Thank you!Thank you!