Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging

F. Schur, W. Hagen, A. De Marco, J. Briggs, Journal of Structural Biology 184 (2013) 394–400.

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Abstract
Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal to noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at which the data was collected, mean that to date the very best structures obtained by sub-tomogram averaging are limited to a resolution of approximately 15. Å. Here, by optimizing data collection and defocus determination steps, we have determined the structure of assembled Mason-Pfizer monkey virus Gag protein using sub-tomogram averaging to a resolution of 8.5. Å. At this resolution alpha-helices can be directly and clearly visualized. These data demonstrate for the first time that high-resolution structural information can be obtained from cryo-electron tomograms using sub-tomogram averaging. Sub-tomogram averaging has the potential to allow detailed studies of unsolved and biologically relevant structures under biologically relevant conditions.
Publishing Year
Date Published
2013-12-01
Journal Title
Journal of Structural Biology
Acknowledgement
The M-PMV ΔPro CANC tubes imaged in this study were a kind gift from Pavel Ulbrich and Tomas Ruml, Institute of Chemical Technology, Prague. The cryo-EM grids were prepared by Tanmay Bharat. This study was technically supported by EMBL’s IT services unit and by Frank Thommen. We thank Martin Schorb and Svetlana Dodonova for discussions and advice; Khanh Huy Bui for advice and scripts to streamline tomogram reconstruction; and Giulia Zanetti, Tanmay Bharat, and Martin Beck for comments on the manuscript. This study was supported by Deutsche Forschungsgemeinschaft grant BR 3635/2-1 to JAGB.
Volume
184
Issue
3
Page
394 - 400
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Schur F, Hagen W, De Marco A, Briggs J. Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging. Journal of Structural Biology. 2013;184(3):394-400. doi:10.1016/j.jsb.2013.10.015
Schur, F., Hagen, W., De Marco, A., & Briggs, J. (2013). Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging. Journal of Structural Biology, 184(3), 394–400. https://doi.org/10.1016/j.jsb.2013.10.015
Schur, Florian, Wim Hagen, Alex De Marco, and John Briggs. “Determination of Protein Structure at 8.5Å Resolution Using Cryo-Electron Tomography and Sub-Tomogram Averaging.” Journal of Structural Biology 184, no. 3 (2013): 394–400. https://doi.org/10.1016/j.jsb.2013.10.015.
F. Schur, W. Hagen, A. De Marco, and J. Briggs, “Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging,” Journal of Structural Biology, vol. 184, no. 3, pp. 394–400, 2013.
Schur F, Hagen W, De Marco A, Briggs J. 2013. Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging. Journal of Structural Biology. 184(3), 394–400.
Schur, Florian, et al. “Determination of Protein Structure at 8.5Å Resolution Using Cryo-Electron Tomography and Sub-Tomogram Averaging.” Journal of Structural Biology, vol. 184, no. 3, Academic Press, 2013, pp. 394–400, doi:10.1016/j.jsb.2013.10.015.

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