Methods Mol Biol. 2024;2715:431-470. doi: 10.1007/978-1-0716-3445-5_27.
ABSTRACT
Structural studies of bio-complexes using single particle cryo-Electron Microscopy (cryo-EM) is nowadays a well-established technique in structural biology and has become competitive with X-ray crystallography. Development of digital registration systems for electron microscopy images and algorithms for the fast and efficient processing of the recorded images and their following analysis has facilitated the determination of structures at near-atomic resolution. The latest advances in EM have enabled the determination of protein complex structures at 1.4-3 Å resolution for an extremely broad range of sizes (from ~100 kDa up to hundreds of MDa (Bartesaghi et al., Science 348(6239):1147-1151, 2015; Herzik et al., Nat Commun 10:1032, 2019; Wu et al., J Struct Biol X 4:100020, 2020; Zhang et al., Nat Commun 10:5511, 2019; Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020; https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year )). In 2022, nearly 1200 structures deposited to the EMDB database were at a resolution of better than 3 Å ( https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year ).To date, the highest resolutions have been achieved for apoferritin, which comprises a homo-oligomer of high point group symmetry (O432) and has rigid organization together with high stability (Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020). It has been used as a test object for the assessments of modern cryo-microscopes and processing methods during the last 5 years. In contrast to apoferritin bacterial secretion systems are typical examples of multi protein complexes exhibiting high flexibility owing to their functions relating to the transportation of small molecules, proteins, and DNA into the extracellular space or target cells. This makes their structural characterization extremely challenging (Barlow, Methods Mol Biol 532:397-411, 2009; Costa et al., Nat Rev Microbiol 13:343-359, 2015). The most feasible approach to reveal their spatial organization and functional modification is cryo-electron microscopy (EM). During the last decade, structural cryo-EM has become broadly used for the analysis of the bio-complexes that comprise multiple components and are not amenable to crystallization (Lyumkis, J Biol Chem 294:5181-5197, 2019; Orlova and Saibil, Methods Enzymol 482:321-341, 2010; Orlova and Saibil, Chem Rev 111(12):7710-7748, 2011).In this review, we will describe the basics of sample preparation for cryo-EM, the principles of digital data collection, and the logistics of image analysis focusing on the common steps required for reconstructions of both small and large biological complexes together with refinement of their structures to nearly atomic resolution. The workflow of processing will be illustrated by examples of EM analysis of Type IV Secretion System.
PMID:37930544 | DOI:10.1007/978-1-0716-3445-5_27
