Cross-linking MS supporting: Structural stabilization of the intrinsically disordered SARS-CoV-2 N by binding to RNA sequences engineered from the viral genome

The nucleocapsid N is one of four structural proteins of the coronaviruses. Its essential role in genome encapsidation makes it a critical therapeutic target for COVID-19 and related diseases. However, the inherent disorder of full-length N has hampered its structural analysis. Here, we describe a stepwise method using viral-derived RNAs to stabilize SARS-CoV-2 N for EM analysis. We identified pieces of RNA from the SARS-CoV-2 genome that promote the formation of structurally homogeneous N dimers, intermediates of assembly, and filamentous capsid-like structures. Building on these results, we engineered a symmetric RNA to stabilize N protein dimers, the building block of high-order assemblies, for EM analyses. We combined domain-specific monoclonal antibodies against N with chemical cross-linking mass spectrometry and cryo-EM to validate the atomic model for the N dimer. The resulting N dimer structure provides insights into its architectural and antigenic principles, which can guide design of pan-coronavirus therapeutics.