Cryo-electron Microscopy of Protein Cages

Abstract

Protein cages are one of the most widely studied objects in the field of cryogenic electron microscopy-encompassing natural and synthetic constructs, from enzymes assisting protein folding such as chaperonin to virus capsids. Tremendous diversity of morphology and function is demonstrated by the structure and role of proteins, some of which are nearly ubiquitous, while others are present in few organisms. Protein cages are often highly symmetrical, which helps improve the resolution obtained by cryo-electron microscopy (cryo-EM). Cryo-EM is the study of vitrified samples using an electron probe to image the subject. A sample is rapidly frozen in a thin layer on a porous grid, attempting to keep the sample as close to a native state as possible. This grid is kept at cryogenic temperatures throughout imaging in an electron microscope. Once image acquisition is complete, a variety of software packages may be employed to carry out analysis and reconstruction of three-dimensional structures from the two-dimensional micrograph images. Cryo-EM can be used on samples that are too large or too heterogeneous to be amenable to other structural biology techniques like NMR or X-ray crystallography. In recent years, advances in both hardware and software have provided significant improvements to the results obtained using cryo-EM, recently demonstrating true atomic resolution from vitrified aqueous samples. Here, we review these advances in cryo-EM, especially in that of protein cages, and introduce several tips for situations we have experienced.

Keywords: 3D reconstruction; Apoferritin; Cryo-electron microscopy; Giant viruses; Image processing; Single particle analysis; Viruses.