Abstract
The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.
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Acknowledgments
This work was supported by Instruct-ULTRA (Coordination and Support Action Number ID 731005) to further develop the services of Instruct-ERIC and iNEXT-Discovery (Grant ID 871037), both funded by the Horizon 2020 program of the European Commission. We acknowledge the support obtained from Thermo Fisher Scientific, Brno.
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Moravcová, J., Dopitová, R., Pinkas, M., Nováček, J. (2021). Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology. 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_15
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DOI: https://doi.org/10.1007/978-1-0716-1406-8_15
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