biostudies-literatureWu XHHS | NIH | National Institute of General Medical SciencesJane Coffin Childs Memorial Fund for Medical ResearchNIGMS NIH HHSNIH HHSe2115001118https://www.ebi.ac.uk/biostudies/studies/S-EPMC8521671biostudies-literatureUnknown118(41)We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). 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. 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 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 Legobody approach thus overcomes the current size limitations of cryo-EM analysis.Proceedings of the National Academy of Sciences of the United States of AmericaCryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).PMC8521671R01GM052586NAP30 GM124165S10 OD021527R01 GM052586Rapoport TAWu XfalseCryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). 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. 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 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 Legobody approach thus overcomes the current size limitations of cryo-EM analysis.2021-01-01T00:00:00Z2021 Oct2026-05-08T15:50:17.674Z2025-02-19T00:03:04.244ZS-EPMC85216713462071610.1073/pnas.2115001118