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Routine sub-2.5 Å cryo-EM structure determination of GPCRs

Author

Listed:
  • Radostin Danev

    (University of Tokyo)

  • Matthew Belousoff

    (Monash University
    Monash University)

  • Yi-Lynn Liang

    (Monash University
    Confo Therapeutics)

  • Xin Zhang

    (Monash University
    Monash University)

  • Fabian Eisenstein

    (University of Tokyo)

  • Denise Wootten

    (Monash University
    Monash University)

  • Patrick M. Sexton

    (Monash University
    Monash University)

Abstract

Cryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Pushing the performance boundaries of the technique requires quantitative knowledge about the contribution of multiple factors. Here, we present an in-depth analysis and optimization of the main experimental parameters in cryo-EM. We combined actual structural studies with methods development to quantify the effects of the Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. By using this information to carefully maximize the experimental performance, it is now possible to routinely determine GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps enables the building of better atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines given here are not limited to GPCRs and can be applied directly to other small proteins.

Suggested Citation

  • Radostin Danev & Matthew Belousoff & Yi-Lynn Liang & Xin Zhang & Fabian Eisenstein & Denise Wootten & Patrick M. Sexton, 2021. "Routine sub-2.5 Å cryo-EM structure determination of GPCRs," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-24650-3
    DOI: 10.1038/s41467-021-24650-3
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    References listed on IDEAS

    as
    1. 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.
    2. Katerina Naydenova & Christopher J. Russo, 2017. "Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy," Nature Communications, Nature, vol. 8(1), pages 1-5, December.
    3. 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.
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    2. Michael W. Martynowycz & Anna Shiriaeva & Max T. B. Clabbers & William J. Nicolas & Sara J. Weaver & Johan Hattne & Tamir Gonen, 2023. "A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    3. Edin Muratspahić & Kristine Deibler & Jianming Han & Nataša Tomašević & Kirtikumar B. Jadhav & Aina-Leonor Olivé-Marti & Nadine Hochrainer & Roland Hellinger & Johannes Koehbach & Jonathan F. Fay & Mo, 2023. "Design and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor," Nature Communications, Nature, vol. 14(1), pages 1-17, December.

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