Affinity Cryo-Electron Microscopy Studies of Viral Particles Captured Directly From Cell Culture

Single particle cryo-electron microscopy (cryo-EM) and 3-D reconstruction have emerged in recent years as a powerful tool for solving high-resolution 3-D structures of viruses and macromolecular complexes. However, sample preparation for single-particle cryo-EM typically relies on a dedicated purification step before EM sample grid preparation, for example, using column chromatography or gradient centrifugation, to obtain homogeneous samples at high concentration. This strategy usually uses large volume of cell culture (hundreds of milli-liters to multiple liters) and takes hours to days to complete. While stable major states of the sample can be successfully prepared, many of the short-lived intermediate states won’t be able to survive the lengthy duration and those minor low abundance states are prone to being ignored. For example, until our attempt with T3 phage [1], tailed dsDNA phage intermediates with incompletely packaged dsDNA genome eluded cryo-EM investigation due to their instability and the faint band visibility in the gradient centrifugation due to low abundance. Therefore, improved sample preparation methods, for example, the affinity cryo-EM grids [2-5], are needed to allow sampling of all states, including the minor, transient intermediates, to fully cover the complete process of the targets for better understanding of structures and functions. Four different affinity grid techniques have been developed for single-particle cryo-EM sample preparation: the Ni–NTA lipid monolayer method [5], the streptavidin 2D crystal-based method [4], the functionalized carbon film method [3], and single-step antibody-based method [2]. However, no structures of subnanometer or higher resolutions using these affinity-grid approaches have been reported and there are concerns if the affinity grids will limit the reconstructions to low resolutions.