Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Fast native-SAD phasing for routine macromolecular structure determination

A Corrigendum to this article was published on 30 June 2015

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

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.

References

  1. Hendrickson, W.A. Q. Rev. Biophys. 47, 49–93 (2014).

    Article  Google Scholar 

  2. Hendrickson, W.A. & Teeter, M.M. Nature 290, 107–113 (1981).

    Article  CAS  Google Scholar 

  3. Mueller, M., Wang, M. & Schulze-Briese, C. Acta Crystallogr. D Biol. Crystallogr. 68, 42–56 (2012).

    Article  CAS  Google Scholar 

  4. Kabsch, W. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  5. de la Fortelle, E. & Bricogne, G. Methods Enzymol. 276, 472–494 (1997).

    Article  CAS  Google Scholar 

  6. Wang, B.C. Methods Enzymol. 115, 90–112 (1985).

    Article  CAS  Google Scholar 

  7. Sheldrick, G.M. Acta Crystallogr. D Biol. Crystallogr. 66, 479–485 (2010).

    Article  CAS  Google Scholar 

  8. Dauter, Z., Dauter, M., de La Fortelle, E., Bricogne, G. & Sheldrick, G.M. J. Mol. Biol. 289, 83–92 (1999).

    Article  CAS  Google Scholar 

  9. Weiss, M.S., Sicker, T., Djinovic-Carugo, K. & Hilgenfeld, R. Acta Crystallogr. D Biol. Crystallogr. 57, 689–695 (2001).

    Article  CAS  Google Scholar 

  10. Liu, Z.J. et al. Acta Crystallogr. A 67, 544–549 (2011).

    Article  CAS  Google Scholar 

  11. Liu, Q. et al. Science 336, 1033–1037 (2012).

    Article  CAS  Google Scholar 

  12. Liu, Q., Liu, Q. & Hendrickson, W.A. Acta Crystallogr. D Biol. Crystallogr. 69, 1314–1332 (2013).

    Article  CAS  Google Scholar 

  13. Li, D. et al. Cryst. Growth Des. 14, 2034–2047 (2014).

    Article  CAS  Google Scholar 

  14. Sharma, A., Kottur, J., Narayanan, N. & Nair, D.T. Nucleic Acids Res. 41, 5104–5114 (2013).

    Article  CAS  Google Scholar 

  15. McCoy, A.J. et al. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  16. Prota, A.E. et al. Science 339, 587–590 (2013).

    Article  CAS  Google Scholar 

  17. Ito, K. et al. J. Mol. Biol. 408, 177–186 (2011).

    Article  CAS  Google Scholar 

  18. Diederichs, K. Acta Crystallogr. D Biol. Crystallogr. 66, 733–740 (2010).

    Article  CAS  Google Scholar 

  19. Zeldin, O.B., Gerstel, M. & Garman, E.F. J. Appl. Crystallogr. 46, 1225–1230 (2013).

    Article  CAS  Google Scholar 

  20. Dinapoli, R. et al. Detect. Assoc. Equip. 731, 68–73 (2013).

    Article  CAS  Google Scholar 

  21. Adams, M.W.W. et al. Acc. Chem. Res. 36, 191–198 (2003).

    Article  CAS  Google Scholar 

  22. Yokoyama, S. et al. Nat. Struct. Biol. 7 (suppl.), 943–945 (2000).

    Article  CAS  Google Scholar 

  23. Basilico, F., Maffini, S., Weir, J. & Prumbaum, D. eLife 3, 1–28 (2014).

    Article  Google Scholar 

  24. Jordan, M.A. & Wilson, L. Nat. Rev. Cancer 4, 253–265 (2004).

    Article  CAS  Google Scholar 

  25. Prota, A.E. et al. J. Cell Biol. 200, 259–270 (2013).

    Article  CAS  Google Scholar 

  26. Henrich, B. et al. Detect. Assoc. Equip. 607, 247–249 (2009).

    Article  CAS  Google Scholar 

  27. Adams, P.D. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  28. Pape, T. & Schneider, T.R. J. Appl. Crystallogr. 37, 843–844 (2004).

    Article  CAS  Google Scholar 

  29. Cowtan, K., Zhang, K. & Main, P. in International Tables for Crystallography Volume F: Crystallography of Biological Macromolecules (eds. Arnold, E., Himmel, D.M. & Rossmann, M.G.) Chapter 15.1, 385–400 (International Union of Crystallography, 2012).

  30. Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  Google Scholar 

  31. Emsley, P. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  32. Murshudov, G.N. et al. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  33. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger LLC <http://www.pymol.org/>.

  34. Cowtan, K. Nautilus software for automated nucleic acid building. CCP4 Newsl. 48 (2012).

Download references

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

Authors and Affiliations

Authors

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.

Ethics declarations

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)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3211

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing