In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography
Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM).
© Springer Science+Business Media, LLC, part of Springer Nature 2021. First Online: 27 December 2020. Cryo-ET work in the Jensen lab is supported by NIH grants R35 GM122588, RO1 A127401, and P50 AI150464 and by the Howard Hughes Medical Institute and performed in the Beckman Institute Resource Center for Transmission Electron Microscopy. M.K. acknowledges a Rubicon postdoctoral fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). We thank Catherine M Oikonomou for reading and editing the manuscript.