A cryo-EM structure of the 2019-nCoV spike protein in the prefusion conformation has been determined at 3.5 Å resolution. The structure reveals a trimer with one of three receptor-binding domains (RBDs) rotated into an accessible conformation. The 2019-nCoV S protein binds ACE2 with higher affinity than SARS-CoV S, suggesting potential for cross-reactivity between antibodies targeting these proteins. However, tested SARS-CoV RBD-specific monoclonal antibodies did not bind to 2019-nCoV S, indicating limited cross-reactivity. The structure provides atomic-level details for vaccine and therapeutic development. The 2019-nCoV S protein is a homotrimer with a metastable prefusion conformation that undergoes structural changes to facilitate membrane fusion. The RBD can adopt "up" or "down" conformations, with "up" being receptor-accessible. The structure shows that the 2019-nCoV S shares structural homology with SARS-CoV S, with a root mean square deviation of 3.8 Å. The RBDs in the "down" conformation differ in positioning between the two proteins. The 2019-nCoV S shares 96% sequence identity with the S protein of the bat coronavirus RaTG13, with notable differences in the S1/S2 protease cleavage site. The S protein of 2019-nCoV uses ACE2 as a receptor, with a binding affinity of ~15 nM, higher than that of SARS-CoV S. The structure also shows that SARS-CoV RBD-specific monoclonal antibodies do not bind to 2019-nCoV S, suggesting limited cross-reactivity. The study highlights the importance of understanding the structure of the spike protein for vaccine and therapeutic development. The structure was determined using cryo-EM and validated with biophysical and structural data. The study provides insights into the structural dynamics of the spike protein and its interaction with the host cell receptor. The results have implications for the development of medical countermeasures against 2019-nCoV.A cryo-EM structure of the 2019-nCoV spike protein in the prefusion conformation has been determined at 3.5 Å resolution. The structure reveals a trimer with one of three receptor-binding domains (RBDs) rotated into an accessible conformation. The 2019-nCoV S protein binds ACE2 with higher affinity than SARS-CoV S, suggesting potential for cross-reactivity between antibodies targeting these proteins. However, tested SARS-CoV RBD-specific monoclonal antibodies did not bind to 2019-nCoV S, indicating limited cross-reactivity. The structure provides atomic-level details for vaccine and therapeutic development. The 2019-nCoV S protein is a homotrimer with a metastable prefusion conformation that undergoes structural changes to facilitate membrane fusion. The RBD can adopt "up" or "down" conformations, with "up" being receptor-accessible. The structure shows that the 2019-nCoV S shares structural homology with SARS-CoV S, with a root mean square deviation of 3.8 Å. The RBDs in the "down" conformation differ in positioning between the two proteins. The 2019-nCoV S shares 96% sequence identity with the S protein of the bat coronavirus RaTG13, with notable differences in the S1/S2 protease cleavage site. The S protein of 2019-nCoV uses ACE2 as a receptor, with a binding affinity of ~15 nM, higher than that of SARS-CoV S. The structure also shows that SARS-CoV RBD-specific monoclonal antibodies do not bind to 2019-nCoV S, suggesting limited cross-reactivity. The study highlights the importance of understanding the structure of the spike protein for vaccine and therapeutic development. The structure was determined using cryo-EM and validated with biophysical and structural data. The study provides insights into the structural dynamics of the spike protein and its interaction with the host cell receptor. The results have implications for the development of medical countermeasures against 2019-nCoV.