Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

Correlative light and electron microscopy is a powerful technique for identification and determination of the structures of interested macromolecules in situ. Combined with sample vitrification, it would be much easier to preserve the native state of macromolecule complexes and distinguish them from the crowded structure environment. In this article, we present a detailed process for the application of the CorrSight system, a light microscope equipped with a cryo module, in combination with a cryo-electron microscope. A relatively long course of up to 7–8 h for cryo module preparation and multichannel light microscopy imaging of vitrified specimen can be sustained. Correlation of light and electron microscopy images at both grid levels to locate squares and square level to locate target particles, and verification of target particles can be performed with the help of AutoEMation software. Cryo-electron tomography is used for obtaining the three-dimensional structure information.

As single particle cryo-electron microscopy has evolved to a new era of atomic resolution, sample heterogeneity still imposes a major limit to the resolution of many macromolecular complexes, especially those with continuous conformational flexibility. Here, we describe a particle segmentation algorithm towards solving structures of molecules composed of several parts that are relatively flexible with each other. In this algorithm, the different parts of a target molecule are segmented from raw images according to their alignment information obtained from a preliminary 3D reconstruction and are subjected to single particle processing in an iterative manner. This algorithm was tested on both simulated and experimental data and showed improvement of 3D reconstruction resolution of each segmented part of the molecule than that of the entire molecule.