Epitope dynamics of SARS-CoV-2 S protein by cryo-electron tomography and single particle cryo-EM

National Institute Of Environmental Health Sciences

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Abstract

We have established high-throughput structure determination workflows using single particle analysis cryo-EM (SPA). These workflows can solve 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 developed a pipeline to determine the structure of S-protein ectodomain from variants in complex with various ligands. We have also deployed 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 are establishing modernized cryo-ET/SVA workflows that make use of the improved quality of DED data and incorporate 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. In collaboration with Dr. Eric Freed at NCI and Dr. Negin Martin at the NIEHS Viral Vector Core we have established a BSL-2 compatible S-protein pseudotyped viral system for expression of wild type and mutant forms of the type I fusion protein. We are applying these approaches to several collaborative projects including: a) characterization of complexes of S with leads identified from nanobody libraries panned with diverse components of S-protein; b) epitope mapping of animal derived polyclonal sera raised against the spike; c) mapping the interaction of S1/S2 with intracellular receptors and epithelial macromolecules; and d) characterization of binding of small molecules and potential therapeutic agents to the spike.