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Fast native-SAD phasing for routine macromolecular structure determination

Nature Methods volume 12, pages 131–133 (2015)Cite this article

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A Corrigendum to this article was published on 30 June 2015

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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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  • 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

Author notes

  1. Katja Bargsten, Deepak T Nair, Federica Basilico & Andreas Boland

    Present address: Present addresses: Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland (K.B.), Regional Centre for Biotechnology, Gurgaon, India (D.T.N.), Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany (F.B.), and Medical Research Council Laboratory of Molecular Biology, Cambridge, UK (A.B.).,

  2. Tobias Weinert, Vincent Olieric and Sandro Waltersperger: These authors contributed equally to this work.

  3. meitian.wang@psi.ch

Authors and Affiliations

  1. Swiss Light Source at Paul Scherrer Institut, Villigen, Switzerland

    Tobias Weinert, Vincent Olieric, Sandro Waltersperger, Ezequiel Panepucci & Meitian Wang

  2. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA

    Lirong Chen, Hua Zhang, Dayong Zhou, John Rose & Bi-Cheng Wang

  3. Laboratory of Biological Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan

    Akio Ebihara

  4. Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan

    Seiki Kuramitsu

  5. Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

    Dianfan Li, Nicole Howe & Martin Caffrey

  6. Boehringer Ingelheim Pharma GmbH and Co. KG, Biberach an der Riss, Germany

    Gisela Schnapp & Alexander Pautsch

  7. Department of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland

    Katja Bargsten, Andrea E Prota & Michel O Steinmetz

  8. National Centre for Biological Sciences, Bangalore, India

    Parag Surana, Jithesh Kottur & Deepak T Nair

  9. Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

    Federica Basilico, Valentina Cecatiello & Sebastiano Pasqualato

  10. Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany

    Andreas Boland & Oliver Weichenrieder

Authors
  1. Tobias Weinert
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Contributions

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.

Corresponding author

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). https://doi.org/10.1038/nmeth.3211

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