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Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis (preprint)
biorxiv; 2021.
Preprint
in English
| bioRxiv | ID: ppzbmed-10.1101.2021.11.17.468963
ABSTRACT
Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO 2 • and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml −1 was sufficient to reconstruct a 3.10 Å resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V 1 -ATPase, β-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 Å resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.
Full text:
Available
Collection:
Preprints
Database:
bioRxiv
Language:
English
Year:
2021
Document Type:
Preprint
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