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Overcoming resolution attenuation during tilted cryo-EM data collection

Author

Listed:
  • Sriram Aiyer

    (Laboratory of Genetics, The Salk Institute for Biological Studies)

  • Philip R. Baldwin

    (Laboratory of Genetics, The Salk Institute for Biological Studies
    Baylor College of Medicine)

  • Shi Min Tan

    (National University of Singapore)

  • Zelin Shan

    (Laboratory of Genetics, The Salk Institute for Biological Studies)

  • Juntaek Oh

    (University of California, San Diego
    Kyung Hee University)

  • Atousa Mehrani

    (Laboratory of Genetics, The Salk Institute for Biological Studies)

  • Marianne E. Bowman

    (Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies)

  • Gordon Louie

    (Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies)

  • Dario Oliveira Passos

    (Laboratory of Genetics, The Salk Institute for Biological Studies)

  • Selena Đorđević-Marquardt

    (Laboratory of Genetics, The Salk Institute for Biological Studies)

  • Mario Mietzsch

    (University of Florida)

  • Joshua A. Hull

    (University of Florida)

  • Shuichi Hoshika

    (Foundation for Applied Molecular Evolution, 13709 Progress Blvd Box 7)

  • Benjamin A. Barad

    (The Scripps Research Institute)

  • Danielle A. Grotjahn

    (The Scripps Research Institute)

  • Robert McKenna

    (University of Florida)

  • Mavis Agbandje-McKenna

    (University of Florida)

  • Steven A. Benner

    (Foundation for Applied Molecular Evolution, 13709 Progress Blvd Box 7)

  • Joseph A. P. Noel

    (Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies
    University of California San Diego)

  • Dong Wang

    (University of California, San Diego
    University of California San Diego
    University of California, San Diego)

  • Yong Zi Tan

    (National University of Singapore
    Disease Intervention Technology Laboratory (DITL), Agency for Science, Technology and Research (A*STAR)
    Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR))

  • Dmitry Lyumkis

    (Laboratory of Genetics, The Salk Institute for Biological Studies
    The Scripps Research Institute
    University of California San Diego)

Abstract

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Suggested Citation

  • Sriram Aiyer & Philip R. Baldwin & Shi Min Tan & Zelin Shan & Juntaek Oh & Atousa Mehrani & Marianne E. Bowman & Gordon Louie & Dario Oliveira Passos & Selena Đorđević-Marquardt & Mario Mietzsch & Jos, 2024. "Overcoming resolution attenuation during tilted cryo-EM data collection," Nature Communications, Nature, vol. 15(1), pages 1-19, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-023-44555-7
    DOI: 10.1038/s41467-023-44555-7
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    References listed on IDEAS

    as
    1. Takanori Nakane & Abhay Kotecha & Andrija Sente & Greg McMullan & Simonas Masiulis & Patricia M. G. E. Brown & Ioana T. Grigoras & Lina Malinauskaite & Tomas Malinauskas & Jonas Miehling & Tomasz Ucha, 2020. "Single-particle cryo-EM at atomic resolution," Nature, Nature, vol. 587(7832), pages 152-156, November.
    2. Anca-Denise Ciută & Kamil Nosol & Julia Kowal & Somnath Mukherjee & Ana S. Ramírez & Bruno Stieger & Anthony A. Kossiakoff & Kaspar P. Locher, 2023. "Structure of human drug transporters OATP1B1 and OATP1B3," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    3. Mark A. Herzik & Mengyu Wu & Gabriel C. Lander, 2019. "High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    4. Kathryn H. Gunn & Saskia B. Neher, 2023. "Structure of dimeric lipoprotein lipase reveals a pore adjacent to the active site," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    5. Ka Man Yip & Niels Fischer & Elham Paknia & Ashwin Chari & Holger Stark, 2020. "Atomic-resolution protein structure determination by cryo-EM," Nature, Nature, vol. 587(7832), pages 157-161, November.
    6. Tristan Bepler & Kotaro Kelley & Alex J. Noble & Bonnie Berger, 2020. "Topaz-Denoise: general deep denoising models for cryoEM and cryoET," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
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