After plunge freezing, protein particles are fixed in a thin vitrified ice layer ( Figure 1A). Third, the grid is rapidly plunged into a liquid cryogen, such as liquid ethane, which has been precooled using liquid nitrogen.

Second, the excess solution is blotted using filter paper, resulting in a thin liquid film spanning across the grid holes. First, a drop of protein solution is applied to a holey carbon film grid that has been pretreated by plasma cleaning. Jacques Dubochet in the early 1980s ( Dubochet et al., 1988) is still widely used. The conventional plunge freezing procedure for cryo-EM sample preparation developed by Dr. However, through cryo-EM studies, researchers have found that the conventional cryo-EM procedure does not always work and that there are obstacles in the specimen preparation procedure. Therefore, cryo-EM has unique advantages and has been successfully applied to the study of the near-atomic resolution structures of challenging protein complexes with high flexibility ( Yan et al., 2015 Plaschka et al., 2017 Ramirez et al., 2019) and small proteins ( Khoshouei et al., 2017 Fan et al., 2019 Zhang et al., 2019 Han et al., 2020 Nygaard et al., 2020). Compared to the X-ray diffraction technique, cryo-EM does not require crystals and only requires a small amount of specimen in its physiological solution. The successful application of direct electron detection devices ( Ruskin et al., 2013 McMullan et al., 2014 Wu et al., 2016) and well-developed imaging processing algorithms ( Scheres, 2012 Li et al., 2013a Punjani et al., 2017 Zheng et al., 2017 Caesar et al., 2020 Punjani et al., 2020 Kimanius et al., 2021 Nakane and Scheres, 2021 Punjani and Fleet, 2021) have greatly improved the resolution of cryoelectron microscopy (cryo-EM), transforming this method into an important approach for determining the structures of biological macromolecules at near-atomic resolution. Here, the various advantages of graphene grids over conventional holey carbon film grids, functionalization of graphene support films, production methods of graphene grids, and origins of pristine graphene contamination are reviewed and discussed. In recent years, grids with support films made of graphene and its derivatives have been developed to efficiently solve these problems. Various problems occur during a vitrification procedure, including poor/nonuniform distribution of particles, preferred orientation of particles, specimen denaturation/degradation, high background from thick ice, and beam-induced motion, which have become important bottlenecks in high-resolution structural studies using cryo-EM in many projects. However, the sample quality using this type of grid is not always ideal for high-resolution imaging even when the specimens in the test tube behave ideally. Specimens are generally prepared in a thin layer of vitrified ice using a holey carbon grid.

3Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, ChinaĬryogenic electron microscopy (cryo-EM) single particle analysis has become one of the major techniques used to study high-resolution 3D structures of biological macromolecules.

2University of Chinese Academy of Sciences, Beijing, China.

1National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

Types of tem grids