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

TLDR

Cryo‑EM is a powerful technique for revealing atomic structures of macromolecules, and new grid designs that preserve thin, uniform vitrified ice and enhance protein adsorption are promising for achieving higher resolution with minimal material and data. The authors aim to develop a method for preparing graphene cryo‑EM grids with up to 99 % monolayer coverage that yields more than 70 % usable grid squares, improving image quality and protein density. Their approach uses only simple, widely available tools, supports customized grid designs, and is broadly compatible with various nanomaterials, enabling easy adoption by most structural biology laboratories. Using these grids, they achieved 2.6‑Å resolution for 52‑kDa streptavidin from 11,000 particles and increased the density of soluble, membrane, and lipoprotein specimens by at least five‑fold, facilitating structural studies of proteins that are difficult to produce in large amounts.

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.