High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy

Abstract

Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids—aimed at preserving thin, uniform vitrified ice and improving protein adsorption—have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with up to 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6-Å resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipoproteins by at least 5-fold, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, this approach supports customized grid designs targeting specific proteins, owing to its broad compatibility with a variety of nanomaterials.

Cryo-EM analysis of a membrane protein embedded in the liposome

“…To produce cryo-samples with evenly distributed proteoliposomes, graphene grids, freshly prepared following our recently reported protocol (39), were used for cryo-sample preparation. With the same sample concentration, the density and distribution of proteoliposomes were evidently improved on graphene grids.…”

Current limitations to high-resolution structure determination by single-particle cryoEM

“…Because graphene is a perfect single crystal, its minimal contribution to background noise can be removed by Fourier filtering if necessary. In principle, these desirable properties make graphene the ideal support film for cryoEM (Han et al ., 2020). The reasons why it has not been used more widely are twofold: (i) it is highly susceptible to surface contamination during production, handling and storage; and (ii) it is extremely hydrophobic.…”

Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

“…As pristine graphene is invisible to the electron beam, it does not raise any additional background and is, therefore, the ideal choice of support layer, especially for small proteins like ABC transporters. The generation of pristine graphene is currently very challenging and limits its broad usage in the field; however, many efforts are put in optimization of graphene grids [84][85][86] and, therefore, the situation is likely to change soon.…”