Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination

Abstract

The application of support layers, such as graphene, to cryo-electron microscopy grids can increase the density of particles imaged, limit particle interactions with the air-water interface, reduce the extent of beam-induced motion, and, in some instances, improve the distribution of particle orientations. This paper describes a robust protocol for coating cryo-EM grids with a monolayer of graphene for improved cryo-sample preparation. Single particle cryo-electron microscopy (cryo-EM) has evolved into a widely used method for visualizing biological macromolecules1. Fueled by advances in direct electron detection2-4, data acquisition5, and image processing algorithms6-10, cryo-EM is now capable of producing near-atomic resolution 3D structures of a fast growing number of macromolecules11. Moreover, by leveraging the single-molecule nature of the approach, users can now determine multiple structures from a single sample12-15, highlighting the promise of using these data to understand heterogeneous structural ensembles16,17. Despite this progress, bottlenecks in cryo-specimen grid preparation persist.