Reconstructing Virus Structures from Nanometer to Near-Atomic Resolutions with Cryo-Electron Microscopy and Tomography

Abstract

The past few decades have seen tremendous advances in single particle electron cryo-microscopy (cryo-EM). The field has matured to the point that near-atomic resolution density maps can be generated for icosahedral viruses without the need for crystallization. In parallel, substantial progress has been made in determining the structures of non-icosahedrally arranged proteins in viruses by employing either single particle cryo-EM or cryo-electron tomography (cryo-ET). Implicit in this course has been the availability of a new generation of electron cryo-microscopes and the development of the computational tools that are essential for generating these maps and models. This methodology has enabled structural biologists to analyze structures in increasing detail for virus particles that are in different morphogenetic and biochemical states. Furthermore, electron imaging of frozen, hydrated cells, in the process of being infected by viruses, has also opened up a new avenue for studying virus structures “in situ”. Here we present the common techniques used to acquire and process cryo-EM and cryo-ET data and discuss their implications for structural virology both now and in the future.

Transmission electron microscopy and the molecular structure of icosahedral viruses

“…A variety of EM imaging approaches can be used to study the structural aspects of icosahedral virus assembly [75,88]. The characteristics of these viruses, including high symmetry and structural homogeneity have placed them at the forefront of structural biology developments.…”

“…In these cases cryo-electron tomography (cryo-ET) can help [75]. Instead of averaging thousands of projections of identical objects lying in different orientations on the grid, cryo-ET combines multiple tilted views of the same field to generate a 3D map of each object.…”

“…After determining the best asymmetric orientation for each masked particle, the complete capsid is reconstructed and used to extract a new mask. The process is iterated to convergence [74,75]. This method was first applied to the study of cyanophage P-SSP7, to show at subnanometer resolution how the 5-to 12-fold symmetry mismatch between capsid and portal is arranged (Fig.…”

Cryo-EM Techniques to Resolve the Structure of HSV-1 Capsid-Associated Components

“…Numerous programs are available to facilitate and automate these processes (28, 34) which have been well reviewed recently and provide a comprehensive background of both theory and application for virus reconstruction (35). …”

“…There are numerous software packages (e.g. SPIDER(39), EMAN (34), IMIRIS (40), EMAN2 (28), MPSA (41), FREALIGN (42), and jalign (43)) that are available to process this data, and their application for processing viruses has been well reviewed previously (35). …”