Letter | Published:

Improved molecular replacement by density- and energy-guided protein structure optimization

Nature volume 473, pages 540543 (26 May 2011) | Download Citation

Abstract

Molecular replacement1,2,3,4 procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods5,6,7,8, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work9,10,11,12,13,14, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

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Acknowledgements

R.J.R., T.C.T. and D.B. thank the NIH (5R01GM092802), the Wellcome Trust (R.J.R.), and HHMI (D.B.) for funding this research. F.D. acknowledges the NIH (P41RR002250) and HHMI. D.F. and A.A. acknowledge support from the Israel Science Foundation. G.O. thanks DK Molecular Enzymology (FWF-project W901) and the Austrian Science Fund (FWF-project P19858). The work of A.W. was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. H.I. acknowledges support from the academy of Finland (1131413). S.M.V. was supported by a grant from the Protein Structure Initiative of National Institute of General Medical Sciences (U54 GM074958). The work of P.R.P. at Argonne National Laboratory was supported by the US Department of Energy’s Office of Science, Biological and Environmental Research GTL programme under contract DE-AC02-06CH11357. We thank all members of the JCSG for their general contributions to the protein production and structural work. The JCSG is supported by the NIH, National Institutes of General Medical Sciences, Protein Structure Initiative (U54 GM094586 and GM074898).

Author information

Affiliations

  1. University of Washington, Department of Biochemistry and HHMI, Seattle, Washington 98195, USA

    • Frank DiMaio
    •  & David Baker
  2. Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA

    • Thomas C. Terwilliger
  3. University of Cambridge, Department of Haematology, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK

    • Randy J. Read
  4. Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA

    • Alexander Wlodawer
  5. Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/3, 8010-Graz, Austria

    • Gustav Oberdorfer
    •  & Ulrike Wagner
  6. University of Cambridge, Department of Biochemistry, Cambridge CB2 1GA, UK

    • Eugene Valkov
  7. Weizmann Institute of Science, Department of Structural Biology, Rehovot 76100, Israel

    • Assaf Alon
    •  & Deborah Fass
  8. Joint Center for Structural Genomics and SSRL, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Herbert L. Axelrod
    •  & Debanu Das
  9. Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027, USA

    • Sergey M. Vorobiev
  10. University of Helsinki, Institute of Biotechnology, FI-00014 Helsinki, Finland

    • Hideo Iwaï
  11. Argonne National Laboratory, Biosciences Division, Argonne, Illinois 60439, USA

    • P. Raj Pokkuluri

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Contributions

F.D., T.C.T., R.J.R. and D.B. developed the methods described in the manuscript; F.D., T.C.T., R.J.R., A.W. and D.B wrote the paper. A.W., G.O., U.W., E.V., A.A., D.F., H.L.A., D.D., S.M.V., H.I. and P.R.P. provided the data and refined one or more structures to completion.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Thomas C. Terwilliger or David Baker.

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    Supplementary Information

    The file contains Supplementary Text, additional references, Supplementary Tables 1-5 and Supplementary Figures 1-3 with legends.

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DOI

https://doi.org/10.1038/nature09964

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