Direct-method SAD phasing

Early attempts

1965 Fan, H. F. Chinese Physics, 1429.1966 Karle, J. Acta Cryst. 21, 273.1970 Hazell, A. C. Nature 227, 269.1973: Sikka, S. K. Acta Cryst. A29, 211.1978: Heinerman, J. J. L., Krabbendam, H., Kroon, J. & Spek, A. L. Acta Cryst. A34, 447.1982 Hauptman, H. A. Acta Cryst. A38, 632.1983: Giacovazzo, C. Acta Cryst. A39, 585.1985: Fan, H. F. and Gu, Y. X. Acta Cryst. A41, 280. 1993: Kyriakidis, C. E., Peschar, R. and Schenk, H. Acta Cryst. A49, 557.1998: Fan, H. F. et al. in Direct Methods for Solving Macromolecular Structures, Kluwer Academic Publishes, pp. 479-485.

Direct-method phasing of the 2 experimental SAD data of the protein aPPAvian Pancreatic PolypeptideSpace group: C2 Unit cell: a=34.18, b=32.92, c=28.44 b=105.3oProtein atoms in ASU: 301Resolution limit: 2.0Anomalous scatterer: Hg, ZnWavelength: 1.542 (Cu-Ka) Df = 7.686, 0.678Phasing: direct methods

Acta Cryst. (1990). A46, 935.

* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USAScience, Vol. 306, Issue 5693, 104-107 (2004)Andrey S. Krasilnikov*, Yinghua Xiao*, Tao Pan, and Alfonso Mondragn*Basis for structural diversity in homologous RNAsFor SAD phasing, the positions of the first 3 Ba+2 sites were identified using SOLVE, 6 more Ba+2 sites were identified and added during heavy atom refinement with SHARP. The phase ambiguity in SAD phasing was partially resolved using OASIS and solvent flattening with SOLOMON as implemented in SHARP. Further improvement of the phases was achieved by doing iterative cycles of phase refinement incorporating phase information frompartially built models followed by solvent flattening.

Difficult SAD phasing SAD phasing at Bijvoet ratio ~ 0.56% An originally unknown protein with 1206 residues in the ASU solved automatically using Cr-Ka sulfur-SAD data

Better initial SAD phases

Two different kinds of initial SAD phases

Comparison on cumulative phase errors sorted in descending order of Fobs60.263.41500058.461.91350056.960.81200062.365.21635255.659.41050054.158.7900052.957. 8750051.257.0600050.056.5450049.157.1300045.857.11500Errors of P+-modified phases ( o )Number of reflectionsErrors of Sim-modified phases ( o )

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

Cu-Ka Fe-SAD302 residues found automaticallyYfbpASpace group: P212121 Unit cell: a = 42.792, b = 54.134, c = 115.222Resolution range: 57.74 1.42Anomalous scatterer: Fe (1)Wavelength: 1.542Df = 3.20/ ~ 1.4%Phased by: OASIS + DM (Cowtan)Automatic model building by: ARP/wARPData provided by Dr. Cheng Yang Rigaku/MSC, USAOASIS-2004 application

Is OASISnecessaryhere?YesIs OASISnecessaryhere?NoWhat would it bewithout using RESOLVE (build only)?

Inside direct-methodSAD phasing

SAD formulation

SAD formulation

P+ formula

Maximizing P(Djh) Djh=b

Replacing Ehexp(ia) with

mhEhexp(iabest)

Fan, H.F. & Gu, Y.X., Acta Cryst. A41, 280-284 (1985)

Fan, H.F. & Gu, Y.X., Acta Cryst. A41, 280-284 (1985)

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