Figure 6.

RCC6 stabilizes the prefusion state independent of RBD orientation. Schematic representation of RCC6 disulfide mutation and expected RBD behavior (modeled from PDB 6VXX and 6VSB) viewed from the “top” (left) and “side” (center) and zoomed in to show the engineered disulfide (right) (A). 2D class averages (top) and 3D reconstruction (bottom) of negative stain EM images of the RCC6_KV designs shows ~45% of the particles maintain all RBDs in the “closed” configuration, while ~55% of particles are partially “open” (B). In contrast, the RCC6_PP design resulted in 47% of particles in the partially “open” state and 53% in a unique “full open” state with no RBDs visible (C). Both RCC6_KV and RCC6_PP had no apparent effect on S2 organization. Similar to RCC3, particles containing the RCC6 design could be identified by negative stain EM images up to 27 days with no apparent degradation (D). Scale bars in (D) indicate 100nm and white circles highlight individual particles.