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A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

Author

Listed:
  • Michael W. Martynowycz

    (University of California
    University of California)

  • Anna Shiriaeva

    (University of California
    University of California)

  • Max T. B. Clabbers

    (University of California
    University of California)

  • William J. Nicolas

    (University of California
    University of California)

  • Sara J. Weaver

    (University of California
    University of California)

  • Johan Hattne

    (University of California
    University of California)

  • Tamir Gonen

    (University of California
    University of California
    University of California)

Abstract

Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.

Suggested Citation

  • 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.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-36733-4
    DOI: 10.1038/s41467-023-36733-4
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    References listed on IDEAS

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    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. Yi-Lynn Liang & Maryam Khoshouei & Mazdak Radjainia & Yan Zhang & Alisa Glukhova & Jeffrey Tarrasch & David M. Thal & Sebastian G. B. Furness & George Christopoulos & Thomas Coudrat & Radostin Danev &, 2017. "Phase-plate cryo-EM structure of a class B GPCR–G-protein complex," Nature, Nature, vol. 546(7656), pages 118-123, June.
    3. Uwe Weierstall & Daniel James & Chong Wang & Thomas A. White & Dingjie Wang & Wei Liu & John C. H. Spence & R. Bruce Doak & Garrett Nelson & Petra Fromme & Raimund Fromme & Ingo Grotjohann & Christoph, 2014. "Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography," Nature Communications, Nature, vol. 5(1), pages 1-6, May.
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