Abstract
Cryo electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.
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Acknowledgments
We thank Jonathan Michalon, Mathieu Schaeffer, Remy Fritz, and Romaric David for IT support. This work was supported by CNRS, Association pour la Recherche sur le Cancer (ARC), Institut National du Cancer (INCa), the Fondation pour la Recherche Médicale (FRM), Ligue nationale contre le cancer (Ligue), Agence National pour la Recherche (ANR), and USIAS (USIAS-2018-012). The electron microscope facility was supported by the Alsace Region, FRM, Inserm, CNRS and ARC, the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01, by Instruct-ERIC and Instruct-ULTRA (Coordination and Support Action Number ID 731005) funded by the EU H2020 framework to further develop the services of Instruct-ERIC.
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von Loeffelholz, O., Klaholz, B.P. (2021). Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection. In: Owens, R.J. (eds) Structural Proteomics. Methods in Molecular Biology, vol 2305. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1406-8_14
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DOI: https://doi.org/10.1007/978-1-0716-1406-8_14
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