biostudies-literatureRobertson MJNIGMS NIH HHS1188-1195https://www.ebi.ac.uk/biostudies/studies/S-EPMC12014012biostudies-literatureUnknown29(12)Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.Nature structural & molecular biologyStructure determination of inactive-state GPCRs with a universal nanobody.PMC12014012R35 GM143061T32 GM089626Seven ABHe FPapasergi-Scott MMChe TSkiniotis GPeroto MCMeyerowitz JGPanova ORobertson MJfalseStructure determination of inactive-state GPCRs with a universal nanobody.Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.2022-01-01T00:00:00Z2022 Dec2026-06-02T00:03:00.213Z2025-07-03T03:05:07.72ZS-EPMC120140123639697910.1038/s41594-022-00859-8