Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Fujita, J.; Makino, F.; Asahara, H.; Moriguchi, M.; Kumano, S.; Anzai, I.; Kishikawa, J.-i.; Matsuura, Y.; Kato, T.; Namba, K.; Inoue, T.

doi:10.1101/2021.11.17.468963

bioRxiv · 10.1101/2021.11.17.468963

Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Fujita, J.·Makino, F.·Asahara, H.·Moriguchi, M.·Kumano, S.·Anzai, I.·Kishikawa, J.-i.·Matsuura, Y.·Kato, T.·Namba, K.·Inoue, T.

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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 ClO2* 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 [A] 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 [A] resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, {beta}-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 [A] 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.

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Fujita, J., Makino, F., Asahara, H., Moriguchi, M., Kumano, S., Anzai, I., Kishikawa, J.-i., Matsuura, Y., Kato, T., Namba, K., Inoue, T.. 2021-11-18. Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis. https://doi.org/10.1101/2021.11.17.468963

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