Epoxidized graphene grid for highly efficient high-resolution cryoEM structural analysis

Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO 2 · as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N 2 atmosphere. Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml −1 was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of β-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules.