We also analyzed the 61 available 2019-nCoV S sequences in the Global Initiative on Sharing All Influenza Data database ( ) and found that there were only nine amino acid substitutions among all deposited sequences. In addition to this insertion of residues in the S1/S2 junction, 29 variant residues exist between 2019-nCoV S and RaTG13 S, with 17 of these positions mapping to the RBD (figs.

In the structure reported here, the S1/S2 junction is in a disordered, solvent-exposed loop. Notably, amino acid insertions that create a polybasic furin site in a related position in hemagglutinin proteins are often found in highly virulent avian and human influenza viruses ( 24). The observation of this phenomenon in 2019-nCoV S suggests that it shares the same mechanism of triggering that is thought to be conserved among the Coronaviridae, wherein receptor binding to exposed RBDs leads to an unstable three-RBD up conformation that results in shedding of S1 and refolding of S2 ( 11, 12).Ģ019-nCoV S shares 98% sequence identity with the S protein from the bat coronavirus RaTG13, with the most notable variation arising from an insertion in the S1/S2 protease cleavage site that results in an “RRAR” furin recognition site in 2019-nCoV ( 19) rather than the single arginine in SARS-CoV (fig.

This seemingly stochastic RBD movement has been captured during structural characterization of the closely related betacoronaviruses SARS-CoV and MERS-CoV, as well as the more distantly related alphacoronavirus porcine epidemic diarrhea virus (PEDV) ( 10, 11, 13, 16). By using the 3D variability feature in cryoSPARC v2 ( 15), we observed breathing of the S1 subunits as the RBD underwent a hinge-like movement, which likely contributed to the relatively poor local resolution of S1 compared with the more stable S2 subunit (movies S1 and S2). Because of the small size of the RBD (~21 kDa), the asymmetry of this conformation was not readily apparent until ab initio 3D reconstruction and classification were performed ( Fig. Because of the indispensable function of the S protein, it represents a target for antibody-mediated neutralization, and characterization of the prefusion S structure would provide atomic-level information to guide vaccine design and development.Īfter collecting and processing 3207 micrograph movies, we obtained a 3.5-Å-resolution three-dimensional (3D) reconstruction of an asymmetrical trimer in which a single RBD was observed in the up conformation. These two states are referred to as the “down” conformation and the “up” conformation, where down corresponds to the receptor-inaccessible state and up corresponds to the receptor-accessible state, which is thought to be less stable ( 10– 13). To engage a host cell receptor, the receptor-binding domain (RBD) of S1 undergoes hinge-like conformational movements that transiently hide or expose the determinants of receptor binding. Receptor binding destabilizes the prefusion trimer, resulting in shedding of the S1 subunit and transition of the S2 subunit to a stable postfusion conformation ( 9). This process is triggered when the S1 subunit binds to a host cell receptor. The S protein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a substantial structural rearrangement to fuse the viral membrane with the host cell membrane ( 7, 8). The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.Ģ019-nCoV makes use of a densely glycosylated spike (S) protein to gain entry into host cells. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo–electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern.

Covid spike