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Cited by 35Advanced Electron Microscopy Techniques and ApplicationsEnzyme Structure and FunctionAdvanced Materials Characterization Techniques

Cryo-electron tomography pipeline for plasma membranes

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Sun, Willy W., Michalak, Dennis J., Sochacki, Kem A., Kunamaneni, Prasanthi, Alfonzo-Méndez, Marco A., Arnold, Andreas M., Strub, Marie-Paule, Hinshaw, Jenny E. +1 more·2025·Nature Communications
DOI 10.1038/s41467-025-56045-zPMC PMC11747107

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Abstract

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with subtomogram averaging and employs a genetically encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cellular environment. The pipeline provides efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of mammalian cells.

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  • Cell unroofing mechanically removes the dorsal surface of adherent cells, exposing the cytoplasmic face of the plasma membrane attached to a solid substrate.

    “Cell unroofing mechanically removes the dorsal surface of adherent cells, thereby exposing the cytoplasmic face of the plasma membrane that remains attached to a solid substrate26,27.”

    From Ghost Neuron preparation unlocks high-resolution structural mapping of intracellular architecture in axons (2026) · cites this paper as [27]

    The Results figure caption (Fig. 1; page unavailable) describes pressurized fluid washing away the apical portions of cells to leave basal plasma membranes on an EM grid, supporting the claim for that preparation. But the same caption also describes unroofing cells in an inverted orientation by washing away their basal portions to isolate apical membranes. Thus, removal of the dorsal surface is not a general description of every unroofing preparation in this paper. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Sun, W. W., Michalak, D. J., Sochacki, K. A., Kunamaneni, P., Alfonzo-Méndez, M. A., Arnold, A. M., Strub, M.-P., Hinshaw, J. E., & Taraska, J. W.
Affiliations
National Institutes of Health (US)
Corresponding author
  • Sochacki, Kem A.
  • Hinshaw, Jenny E.
  • Taraska, Justin W.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-01-20
Volume
16
Issue
1
Pages
855-855
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-56045-z
PMC
PMC11747107
OpenAlex
W4406602229
Cited by
35
References
2
Citations by year
2026: 20 · 2025: 12 · 2024: 3
FWCI
10.44
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-sa)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health and Human Services (ZIAHL006098, ZIADK060100)
  • Silicon Valley Community Foundation
  • University of Pittsburgh
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (ZIAHL006098)
  • National Institute of Diabetes and Digestive and Kidney Diseases (ZIADK060100)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Physical Sciences › Engineering › Biomedical Engineering › Advanced Materials Characterization Techniques
Keywords
cryo-electron tomography, plasma membrane, sample preparation, subtomogram averaging, membrane protein structure, mammalian cells

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