Fast native-SAD phasing for routine macromolecular structure determination

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  • A Corrigendum to this article was published on 30 June 2015

Abstract

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

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Figure 1: Eleven structures solved by native-SAD phasing.
Figure 2: Molecular weight versus number of anomalous scattering atoms of all SAD-solved structures.

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Change history

  • 06 February 2015

    In the version of this article initially published, the Hendrickson formula in the Figure 2 legend incorrectly had (2NA/NP)1/2 divided by (f″/Zeff); these terms should have been multiplied. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors would like to thank C. Schulze-Briese, W. Glettig, M. Salathe, X. Wang and C. Pradervand for developing the PRIGo goniometer and C. Dekker for her help in validating the data collection method.

Author information

M.W. and B.-C.W. conceived the research; S.W., V.O. and M.W. designed the experiments; L.C., H.Z., D.Z., J.R., A.E., S.K., D.L., N.H., G.S., A.P., K.B., A.E.P., P.S., J.K., D.T.N., F.B., V.C., S.P., A.B. and O.W. prepared samples; T.W., S.W., V.O., E.P. and M.W. performed experiments; T.W., S.W., V.O. and M.W. analyzed data; T.W., V.O., M.O.S., M.C. and M.W. wrote the manuscript with contributions from all authors.

Correspondence to Meitian Wang.

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Competing interests

G.S. and A.P. declare competing financial interests as employees of Boehringer Ingelheim Pharma GmbH & Co. KG.

Integrated supplementary information

Supplementary Figure 1 Substructure and hand determination with SHELXD/E.

Supplementary Figure 2 Analysis of anomalous peak heights.

Anomalous peak heights are shown as connected dots with data sets merged from consecutive 360° “turns” (see Supplementary Table 3 for details). Source data

Supplementary Figure 3 Representative sections of experimental phased maps contoured at 1σ.

Supplementary Figure 4 Comparison of anomalous peak heights between conventional, high-redundancy single-orientation and high-redundancy multiple-orientation T2R-TTL data sets.

Low redundancy single orientation (1 × 360º at Chi = 0º), high redundancy single orientation (8 × 360º at Chi = 0º) and high redundancy multiple orientation (1 × 360º at Chi = 0º, 5º, 10º, 15º, 20º, 25º, 30º and 0°) T2R-TTL data sets were measured with similar total X-ray dose. All data were collected on one crystal. The anomalous peak heights calculated to 3.0 Å resolution are substantially higher for the low dose high redundancy multiple orientation data collection protocol. Source data

Supplementary Figure 5 Ideal exposure for a phasing experiment with T2R-TTL as a test case.

Data sets of 180º were collected at 6 keV with 1.5 × 1010 photons/s with the following oscillation range / exposure time: (a) - 0.1° / 0.025 s; (b) - 0.1° / 0.1 s; (c) - 0.1° / 0.4 s. Data were processed with XDS, and the plots were generated from XDS_ASCII.HKL. Ideal exposure was characterized by strong reflections in the overall count range from 1000 to 10000 reaching the final (I/σ)asymptotic value (marked with dotted lines). These reflections were measured as accurately as possible without unnecessary X-ray exposure, thus optimally balancing overall radiation damage with redundancy.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 3118 kb)

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Weinert, T., Olieric, V., Waltersperger, S. et al. Fast native-SAD phasing for routine macromolecular structure determination. Nat Methods 12, 131–133 (2015) doi:10.1038/nmeth.3211

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