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Abstract
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 ClO2· as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N2 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.
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1 Osaka University, Graduate School of Frontier Biosciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, JEOL YOKOGUSHI Research Alliance Laboratories, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, Graduate School of Pharmaceutical Sciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971)
2 Osaka University, Graduate School of Frontier Biosciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, JEOL YOKOGUSHI Research Alliance Laboratories, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); JEOL Ltd, Akishima, Japan (GRID:grid.410892.6) (ISNI:0000 0001 2284 8430)
3 Osaka University, Graduate School of Pharmaceutical Sciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, Open and Transdisciplinary Research Initiatives, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971)
4 Osaka University, Graduate School of Pharmaceutical Sciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971)
5 Osaka University, Department of Molecular Virology, Research Institute for Microbial Diseases, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971)
6 Osaka University, Institute for Protein Research, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Kyoto Sangyo University, Department of Molecular Biosciences, Kita-ku, Japan (GRID:grid.258798.9) (ISNI:0000 0001 0674 6688)
7 Osaka University, Center for Infectious Disease Education and Research, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, Laboratory of Virus Control, Research Institute for Microbial Diseases, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971)
8 Osaka University, Institute for Protein Research, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971)
9 Osaka University, Graduate School of Frontier Biosciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, JEOL YOKOGUSHI Research Alliance Laboratories, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); RIKEN Center for Biosystems Dynamics Research and SPring-8 Center, Suita, Japan (GRID:grid.508743.d)
10 Osaka University, Graduate School of Pharmaceutical Sciences, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); Osaka University, Open and Transdisciplinary Research Initiatives, Suita, Japan (GRID:grid.136593.b) (ISNI:0000 0004 0373 3971); dotAqua Inc., Suita, Japan (GRID:grid.136593.b)