Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100–250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2–3 d, allowing for several pause points during the procedure.
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Cryo-ET representative tomograms have been deposited in the Electron Microscopy Data Bank under the accession code EMD-21039.
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We thank Julia Mahamid, Bernd Fruhberger and Villa lab members for insightful discussions and technical support. The subtomogram average of ribosome shown in Fig. 8 was performed by Robert Buschauer. This work was supported by an NIH Director’s New Innovator Award 1DP2GM123494-01 and the National Science Foundation MRI grant NSF DBI 1920374. We acknowledge the use of the UC San Diego cryo-Electron Microscopy Facility (partially supported by a gift from the Agouron Institute to UC San Diego) and the San Diego Nanotechnology Infrastructure of UC San Diego, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (ECCS-1542148). D.S. is supported by the Damon Runyon Cancer Research Foundation (DRG-#2364-19).
F.R.W., R.W., D.S., M.S., J.P. and E.V. have no competing interests. R.S. and H.P. are employees of TFS, and P.F. was an employee.
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Wagner, F.R., Watanabe, R., Schampers, R. et al. Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography. Nat Protoc 15, 2041–2070 (2020). https://doi.org/10.1038/s41596-020-0320-x
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