{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Wu X"],"funding":["HHS | NIH | National Institute of General Medical Sciences","Jane Coffin Childs Memorial Fund for Medical Research","NIGMS NIH HHS","NIH HHS"],"pagination":["e2115001118"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC8521671"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["118(41)"],"pubmed_abstract":["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."],"journal":["Proceedings of the National Academy of Sciences of the United States of America"],"pubmed_title":["Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)."],"pmcid":["PMC8521671"],"funding_grant_id":["R01GM052586","NA","P30 GM124165","S10 OD021527","R01 GM052586"],"pubmed_authors":["Rapoport TA","Wu X"],"additional_accession":[]},"is_claimable":false,"name":"Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).","description":"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.","dates":{"release":"2021-01-01T00:00:00Z","publication":"2021 Oct","modification":"2026-05-08T15:50:17.674Z","creation":"2025-02-19T00:03:04.244Z"},"accession":"S-EPMC8521671","cross_references":{"pubmed":["34620716"],"doi":["10.1073/pnas.2115001118"]}}