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

Author

Listed:
  • Ricardo M. Sanchez

    (Max Planck Institute for Biophysics
    Goethe University of Frankfurt am Main)

  • Yingyi Zhang

    (Max Planck Institute for Biophysics
    Goethe University of Frankfurt am Main)

  • Wenbo Chen

    (Max Planck Institute for Biophysics
    Goethe University of Frankfurt am Main)

  • Lea Dietrich

    (Max Planck Institute for Biophysics)

  • Mikhail Kudryashev

    (Max Planck Institute for Biophysics
    Goethe University of Frankfurt am Main)

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.

Suggested Citation

  • Ricardo M. Sanchez & Yingyi Zhang & Wenbo Chen & Lea Dietrich & Mikhail Kudryashev, 2020. "Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM," Nature Communications, Nature, vol. 11(1), pages 1-7, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17466-0
    DOI: 10.1038/s41467-020-17466-0
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    Cited by:

    1. Nikita Balyschew & Artsemi Yushkevich & Vasilii Mikirtumov & Ricardo M. Sanchez & Thiemo Sprink & Mikhail Kudryashev, 2023. "Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    2. Yanan Zhu & Christopher W. Koo & C. Keith Cassidy & Matthew C. Spink & Tao Ni & Laura C. Zanetti-Domingues & Benji Bateman & Marisa L. Martin-Fernandez & Juan Shen & Yuewen Sheng & Yun Song & Zhengyi , 2022. "Structure and activity of particulate methane monooxygenase arrays in methanotrophs," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    3. Shouwen Du & Ruchao Peng & Wang Xu & Xiaoyun Qu & Yuhang Wang & Jiamin Wang & Letian Li & Mingyao Tian & Yudong Guan & Jigang Wang & Guoqing Wang & Hao Li & Lingcong Deng & Xiaoshuang Shi & Yidan Ma &, 2023. "Cryo-EM structure of severe fever with thrombocytopenia syndrome virus," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    4. Kotaro Kelley & Ashleigh M. Raczkowski & Oleg Klykov & Pattana Jaroenlak & Daija Bobe & Mykhailo Kopylov & Edward T. Eng & Gira Bhabha & Clinton S. Potter & Bridget Carragher & Alex J. Noble, 2022. "Waffle Method: A general and flexible approach for improving throughput in FIB-milling," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    5. Jing Cheng & Tong Liu & Xin You & Fa Zhang & Sen-Fang Sui & Xiaohua Wan & Xinzheng Zhang, 2023. "Determining protein structures in cellular lamella at pseudo-atomic resolution by GisSPA," Nature Communications, Nature, vol. 14(1), pages 1-9, December.

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