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Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM

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Sanchez,  Ricardo M.
Sofja Kovalevskaja Group, Max Planck Institute of Biophysics, Max Planck Society;
Buchmann Institute for Molecular Life Sciences, Goethe-University of Frankfurt, 60348 Frankfurt am Main, Germany;

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Zhang,  Yingyi
Sofja Kovalevskaja Group, Max Planck Institute of Biophysics, Max Planck Society;
Buchmann Institute for Molecular Life Sciences, Goethe-University of Frankfurt, 60348 Frankfurt am Main, Germany;

/persons/resource/persons238643

Chen,  Wenbo
Sofja Kovalevskaja Group, Max Planck Institute of Biophysics, Max Planck Society;
Buchmann Institute for Molecular Life Sciences, Goethe-University of Frankfurt, 60348 Frankfurt am Main, Germany;

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Dietrich,  Lea
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Kudryashev,  Misha
Sofja Kovalevskaja Group, Max Planck Institute of Biophysics, Max Planck Society;
Buchmann Institute for Molecular Life Sciences, Goethe-University of Frankfurt, 60348 Frankfurt am Main, Germany;

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Citation

Sanchez, R. M., Zhang, Y., Chen, W., Dietrich, L., & Kudryashev, M. (2020). Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM. Nature Communications, 11: 3709. doi:10.1038/s41467-020-17466-0.


Cite as: https://hdl.handle.net/21.11116/0000-0006-C514-5
Abstract
Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.