Epitope dynamics of native SARSCoV-2 S protein by cryo-electron tomography

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Principal Investigator: William  Copeland
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2020
Award: $196,477
Funding agency: National Institute of Environmental Health Sciences

Deployment of a SPA pipeline to determine the structure of S-protein soluble ectodomain and its complexes: We have established high-throughput structure determination workflows using single particle analysis cryo-EM (SPA). These workflows are capable of solving the structure of macromolecular complexes at near atomic resolution in less than 24 hours. In collaboration with Dr. Robert Petrovich at the Protein Expression and Purification Facility, we establish a pipeline to determine the structure of S-protein ectodomain in complex with various ligands. Meanwhile, our collaborator Dr. Priyamvada Acharya at DHVI established a stably closed trimeric ectodomain. 
Deployment of a cryo-electron tomography (cryo-ET) and sub-volume averaging (SVA) pipeline to determine the structure of full length (FL) S-protein and its complexes in the context of the viral envelope: In collaboration with Dr. Alberto Bartesaghi at Duke, we had previously established a modernized cryo-ET/SVA workflow that makes use of the improved quality of DED data and incorporates novel image processing techniques to obtain high-resolution tomographic reconstructions, identify objects of interest in a crowded environment and determine their near-atomic resolution structure using advanced methods for sub-volume averaging. In collaboration with Dr. Eric Freed at NCI and Dr. Negin Martin at the NIEHS Viral Vector Core we have identified a BSL-2 compatible S-protein pseudotyped viral system for expression of wild type and mutant forms. 
Using these workflows in SPA and cryoET/SVA, we are working on the following projects: a) Structure of complexes of S-protein with nanobodies in collaboration with Dr. Matthew Hall at NCATS; b) Mapping the binding of potential therapeutic agents with S-protein in collaborations with Drs. Lars Pedersen, Stavros Garantziotis and Nicole Kleinstreuer at NIEHS; c) Determining the effect of insect proteins on viral particles in collaboration with Dr. Geoffrey Mueller.

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