Researchers have determined two cryo-electron microscopy structures of the SARS-CoV-2 spike (S) protein: one in the prefusion conformation (2.9 Å resolution) and one in the postfusion conformation (3.0 Å resolution). The prefusion trimer has three receptor-binding domains (RBDs) clamped by a segment adjacent to the fusion peptide. The postfusion structure is decorated with N-linked glycans, suggesting possible protective roles against immune responses and harsh conditions. These findings enhance understanding of SARS-CoV-2 entry and may guide vaccine and therapeutic development. The S protein is a heavily glycosylated type I membrane protein that induces neutralizing antibodies and is a key target for vaccine development. It is first produced as a precursor that trimerizes and is cleaved by proteases into S1 and S2 fragments. Binding of S1 to ACE2 and proteolytic cleavage of S2 trigger dissociation of S1 and refolding of S2 into a postfusion conformation, forming a trimeric hairpin structure. The study shows that the prefusion S trimer has an ordered N terminus with a disulfide bond and an N-linked glycan. A segment downstream of the fusion peptide, designated FPPR, is ordered in the full-length structure but disordered in the stabilized soluble S trimer. The FPPR may play a role in clamping down the RBD and stabilizing the closed conformation of the S trimer. The postfusion S2 trimer has a rigid structure with N-linked glycans along its long axis. The structure reveals that the S2 trimer forms a six-helix bundle and that the postfusion conformation may have a protective role by inducing non-neutralizing antibody responses and shielding the more vulnerable prefusion S1/S2 trimers. The study highlights the importance of understanding the structural dynamics of the S protein for vaccine development. Vaccines using the full-length wild-type S protein may produce immunodominant, non-neutralizing epitopes. Stabilizing the prefusion conformation with proline mutations may not be optimal as it could lead to a relaxed apex that induces antibodies that do not efficiently recognize S trimer spikes on the virus. The findings suggest that the postfusion S2 trimer may be present on infectious virions and that vaccines using inactivated viruses may require additional quality control tests. Structure-guided immunogen design is critical for future vaccine development, especially if SARS-CoV-2 becomes seasonal and undergoes antigenic drift.Researchers have determined two cryo-electron microscopy structures of the SARS-CoV-2 spike (S) protein: one in the prefusion conformation (2.9 Å resolution) and one in the postfusion conformation (3.0 Å resolution). The prefusion trimer has three receptor-binding domains (RBDs) clamped by a segment adjacent to the fusion peptide. The postfusion structure is decorated with N-linked glycans, suggesting possible protective roles against immune responses and harsh conditions. These findings enhance understanding of SARS-CoV-2 entry and may guide vaccine and therapeutic development. The S protein is a heavily glycosylated type I membrane protein that induces neutralizing antibodies and is a key target for vaccine development. It is first produced as a precursor that trimerizes and is cleaved by proteases into S1 and S2 fragments. Binding of S1 to ACE2 and proteolytic cleavage of S2 trigger dissociation of S1 and refolding of S2 into a postfusion conformation, forming a trimeric hairpin structure. The study shows that the prefusion S trimer has an ordered N terminus with a disulfide bond and an N-linked glycan. A segment downstream of the fusion peptide, designated FPPR, is ordered in the full-length structure but disordered in the stabilized soluble S trimer. The FPPR may play a role in clamping down the RBD and stabilizing the closed conformation of the S trimer. The postfusion S2 trimer has a rigid structure with N-linked glycans along its long axis. The structure reveals that the S2 trimer forms a six-helix bundle and that the postfusion conformation may have a protective role by inducing non-neutralizing antibody responses and shielding the more vulnerable prefusion S1/S2 trimers. The study highlights the importance of understanding the structural dynamics of the S protein for vaccine development. Vaccines using the full-length wild-type S protein may produce immunodominant, non-neutralizing epitopes. Stabilizing the prefusion conformation with proline mutations may not be optimal as it could lead to a relaxed apex that induces antibodies that do not efficiently recognize S trimer spikes on the virus. The findings suggest that the postfusion S2 trimer may be present on infectious virions and that vaccines using inactivated viruses may require additional quality control tests. Structure-guided immunogen design is critical for future vaccine development, especially if SARS-CoV-2 becomes seasonal and undergoes antigenic drift.