Defocus corrected large area cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections
- University of Massachusetts, Howard Hughes Medical Institute,, United States
- Harvard University, United States
Abstract
A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study we use a new data-acquisition scheme called 'Defocus-Corrected Large-Area cryo-EM' (DeCo-LACE) to collect high-resolution images of entire sections (100 - 250 nm thick lamellae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide 'maps' of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.
Data availability
Cryo-EM movies, motion-corrected images and 2DTM results have been deposited in EMPIAR under accession code EMPIAR-11063. The custom cisTEM version is available under https://github.com/jojoelfe/cisTEM/tree/2574dbdf6161658fd177660b3a841100a792f61b until features have been integrated into a cisTEM release. The ChimeraX extension for rendering is available under https://github.com/jojoelfe/tempest. This manuscript was prepared using the manubot package [37] . The corresponding repository containing all scripts used for figure generation can be found under https://github.com/jojoelfe/deco_lace_template_matching_manuscript.
Article and author information
Author details
Nikolaus Grigorieff
Funding
Howard Hughes Medical Institute (HHMI Investigator)
- Nikolaus Grigorieff
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Elferich et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Defocus corrected large area cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections
eLife 11:e80980.
https://doi.org/10.7554/eLife.80980
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