Structural analysis of protein complexes by cryo electron microscopy

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3–5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147–1151, 2051; Bai et al., Nature 525(7568):212–217, 2015; Vinothkumar et al., Nature 515(7525):80–84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265–273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System

Preparation of proteins and macromolecular assemblies for cryo-electron microscopy

Cryo-electron microscopy has become popular as the penultimate step on the road to structure determination for many proteins and macromolecular assemblies. The process of obtaining high-resolution images of a purified biomolecular complex in an electron microscope often follows a long, and in many cases exhaustive screening process in which many iterative rounds of protein purification are employed and the sample preparation procedure progressively re-evaluated in order to improve the distribution of particles visualized under the electron microscope, and thus\ua0maximize the opportunity for high-resolution structure determination. Typically, negative stain electron microscopy is employed to obtain a preliminary assessment of the sample quality, followed by cryo-EM which first requires the identification of optimal vitrification conditions. The original methods for frozen-hydrated specimen preparation developed over 40\ua0years ago still enjoy widespread use today, although recent developments have set the scene for a future where more systematic and high-throughput approaches to the preparation of vitrified biomolecular complexes may be routinely employed. Here we summarize current approaches and ongoing innovations for the preparation of frozen-hydrated single particle specimens for cryo-EM, highlighting some of the commonly encountered problems and approaches that may help overcome these