Info box 4: Single-particle electron cryo-microscopy and electron cryo-tomography

In single-particle electron cryo-microscopy (cryoEM), the purified protein or protein complex is preserved in vitreous water on sample grids allowing for their native structural state to be maintained [26]. Imaging of such samples is performed under cryogenic temperatures to protect the specimen from radiation damage. Here, the assumption is that the particles studied obtain random orientations on the sample grid. During imaging, tens of thousands up to million(s) of two-dimensional (2D) projections of individual particles are collected [56, 57]. These 2D projections are aligned and averaged to generate a three-dimensional (3D) reconstruction of the protein or protein complex using dedicated image-processing algorithms [58, 59]. In cases where the 3D structure reaches atomic resolution, the amino acid sequence can be built into the 3D map to generate a 3D model of the protein or protein complex. Single-particle cryoEM is typically applied for macromolecular complexes ranging in size from below 100 kDa (such as hemoglobin [60]) to MDa (such as intact viruses [61]).

In contrast, electron cryo-tomography (cryoET) allows for the three-dimensional visualization of intact cells and cellular structures. The sample(s) studied (intact cells, larger viruses) is preserved in vitreous water on special sample grids allowing for its native structural state to be maintained, much like in single-particle cryoEM. However, during imaging, the sample is rotated within the microscope by tilting the grid along one; sometimes two axes, and a ‘tilt-series’ of two-dimensional (2D) projections are acquired and then used for the calculation of a three-dimensional (3D) reconstruction or tomogram [62]. Due to this imaging technique and the lack of single-particle averaging for a higher signal–noise ratio, the achievable resolution is limited. The resolution is further limited by the thickness of the sample, as the electron beam typically can only penetrate 500 nm into the sample [62]. This means in practice that only prokaryotes can be imaged in toto, whereas other cells must be thinned down [63]. Mammalian cells and tissues are often sliced into thinner sections via cryo-sectioning or focused ion beam milling before visualization [25, 64]. The resolution of certain symmetric and repetitive features in the tomogram—such as smaller cellular components or viral surface proteins—can be increased by sub-tomogram averaging [65, 66]. Here, these features are processed as individual protein(s) much like as in single-particle cryoEM, where 2D projections of individual particles are collected, aligned and averaged to generate a 3D reconstruction of the protein or protein complex, with the distinction that in sub-tomogram averaging, the particles are represented by 3D volumes rather than 2D projections.