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GraFix: sample preparation for single-particle electron cryomicroscopy

Nature Methods volume 5, pages 53–55 (2008)Cite this article

Abstract

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.

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Figure 1: Schematics of the GraFix setup.
Figure 2: Effect of GraFix-based sample preparation on B complex spliceosomes and the 70S ribosome.
Figure 3: Improved structure of GraFix-prepared APC/C.

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Acknowledgements

We thank H. Kohansal, T. Conrad and W. Jahn for expert technical assistance, U. Göringer and M. Brecht for preparing the RNA editing complex, M. Rodnina and K. Gromadski for preparing the 70S ribosome complex, and C. Will for preparing the 17S U2 snRNP. The work was supported by grants from the Federal Ministry of Education and Research, Germany and from the Sixth Framework Programme of the European Union via 3DRepertoire (to H.S.). Work in the laboratory of J.M.P. is supported by Boehringer Ingelheim and by Spots of Excellence of the city of Vienna. N.F. is supported by a Boehringer-Ingelheim fellowship. E.W. is supported by a 'Studienstiftung des deutschen Volkes' fellowship.

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Authors and Affiliations

  1. Department of Cellular Biochemsitry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Berthold Kastner, Klaus Hartmuth, Jochen Deckert, Elmar Wolf & Reinhard Lührmann

  2. 3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Niels Fischer, Monika Mariola Golas, Bjoern Sander, Prakash Dube, Daniel Boehringer, Florian Hauer, Hannes Uchtenhagen & Holger Stark

  3. Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Henning Urlaub

  4. Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Dietmar Poerschke

  5. Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, Vienna, 1030, Austria

    Franz Herzog & Jan Michael Peters

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  1. Berthold Kastner
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Correspondence to Berthold Kastner or Holger Stark.

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Max Planck Innovation has files a patent on this method.

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Supplementary Figures 1–3, Supplementary Data, Supplementary Methods (PDF 1220 kb)

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Kastner, B., Fischer, N., Golas, M. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat Methods 5, 53–55 (2008). https://doi.org/10.1038/nmeth1139

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