No abstract

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded (down) to an exposed (up) state in order to bind the human ACE2 receptor and infect cells. While snapshots of the up and down states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 200 μs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen activating protein has been exploited to produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and superresolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved using physiological buffers and cellular media, and does not require additives or switching buffer to obtain superresolution images. We evaluate superresolution properties and images obtained from a selected fluorogen activating protein clone fused to actin, and show that the photon counts per object fall between those typically reported for fluorescent proteins and switching dye-pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.