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High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM

Author

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  • Mark A. Herzik

    (The Scripps Research Institute
    University of California, San Diego)

  • Mengyu Wu

    (The Scripps Research Institute)

  • Gabriel C. Lander

    (The Scripps Research Institute)

Abstract

Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.

Suggested Citation

  • 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.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-08991-8
    DOI: 10.1038/s41467-019-08991-8
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    Cited by:

    1. 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.
    2. 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.
    3. Katsuya Takahashi & Yongchan Lee & Angela Fago & Naim M. Bautista & Jay F. Storz & Akihiro Kawamoto & Genji Kurisu & Tomohiro Nishizawa & Jeremy R. H. Tame, 2024. "The unique allosteric property of crocodilian haemoglobin elucidated by cryo-EM," Nature Communications, Nature, vol. 15(1), pages 1-10, December.
    4. Marta Ukleja & Lara Kricks & Gabriel Torrens & Ilaria Peschiera & Ines Rodrigues-Lopes & Marcin Krupka & Julia García-Fernández & Roberto Melero & Rosa Campo & Ana Eulalio & André Mateus & María López, 2024. "Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains," Nature Communications, Nature, vol. 15(1), pages 1-21, December.

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