Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Moreover, a 3.4 Å cryoelectron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 more » receptor binding. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1 A and RBD epitopes on SARS-CoV-2 spike. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/ neutralization of IgGs over Fabs. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Differences in sybody recognition of several recently identified RBD variants are explained by these structures. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both “up” and “down” configurations, but others may not. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. In contrast, Sb68 more » interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. The Legobody approach thus overcomes the current size limitations of cryo-EM = ,Ĭombating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The overall ensemble of ~120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing.

The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM).

Rigid protein scaffold