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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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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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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DOI: https://doi.org/10.1038/nmeth1139
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