COVID-19, caused by SARS-CoV-2, has devasted global health and economics. Vaccines are being deployed worldwide to gain control of the pandemic, although emergence of fast-spreading “variants of concern” (VOCs) have caused concern. Mutations in the spike (S) protein are under scrutiny due to its essential role in the virus life cycle, and being the dominant target of neutralizing antibodies. Widespread vaccine hesitancy and the current spread of the Delta variant provide fertile ground for emergence of vaccine resistant variants. We and others have shown that variants use a plethora of strategies to modify antibody and receptor interactive surfaces, and spike conformation, resulting in antibody evasion and greater infectivity. Over the last two years, utilizing urgent supplement funding from the NIH, we studied the structures of SARSCoV-2 S proteins and have established workflows spanning structure, biochemistry, biophysics and computation. Here we propose to continue the essential work of detangling the effects of variant S protein mutations, and to enhance our understanding of spike structure to further efforts to predict where the virus is heading and to inform novel vaccine designs. The scientific premise of this grant is that understanding spike structure and allostery will provide insights into its function, inform vaccine development, and provide mechanistic information essential for relating spike structure to beta-CoV replication, evolution, and immune evasion. The innovations in this grant derive from technologies we have developed for structural analyses of the S protein: an integrative structural biology pipeline combines cryo-electron microscopy (cryo-EM), Negative Stain Electron Microscopy (NSEM) and X-ray crystallography, with computational methods, and biochemical and biophysical analyses to study structural and functional properties of the spike, including furin cleavage, receptor binding, and antigenicity.