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- PDB-7ul2: CryoEM Structure of Inactive NTSR1 Bound to SR48692 and Nb6 -

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Basic information

Entry
Database: PDB / ID: 7ul2
TitleCryoEM Structure of Inactive NTSR1 Bound to SR48692 and Nb6
Components
  • Nanobody 6
  • Neurotensin receptor 1
KeywordsMEMBRANE PROTEIN / Antagonist / Complex
Function / homology
Function and homology information


response to acrylamide / adenylate cyclase-inhibiting opioid receptor signaling pathway / dynorphin receptor activity / regulation of saliva secretion / negative regulation of luteinizing hormone secretion / sensory perception of temperature stimulus / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / symmetric synapse / positive regulation of eating behavior ...response to acrylamide / adenylate cyclase-inhibiting opioid receptor signaling pathway / dynorphin receptor activity / regulation of saliva secretion / negative regulation of luteinizing hormone secretion / sensory perception of temperature stimulus / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / symmetric synapse / positive regulation of eating behavior / G protein-coupled opioid receptor activity / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / G protein-coupled opioid receptor signaling pathway / regulation of membrane depolarization / positive regulation of dopamine secretion / positive regulation of arachidonate secretion / L-glutamate import across plasma membrane / sensory perception / regulation of respiratory gaseous exchange / maternal behavior / positive regulation of potassium ion transmembrane transport / receptor serine/threonine kinase binding / positive regulation of inhibitory postsynaptic potential / negative regulation of systemic arterial blood pressure / neuropeptide binding / positive regulation of p38MAPK cascade / negative regulation of release of sequestered calcium ion into cytosol / positive regulation of glutamate secretion / eating behavior / temperature homeostasis / response to lipid / positive regulation of inositol phosphate biosynthetic process / detection of temperature stimulus involved in sensory perception of pain / conditioned place preference / neuropeptide signaling pathway / estrous cycle / MECP2 regulates neuronal receptors and channels / behavioral response to cocaine / axon terminus / sensory perception of pain / T-tubule / Peptide ligand-binding receptors / sarcoplasmic reticulum / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / adult locomotory behavior / learning / response to nicotine / locomotory behavior / cellular response to glucose stimulus / G protein-coupled receptor activity / response to insulin / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / response to estrogen / cytoplasmic side of plasma membrane / terminal bouton / synaptic vesicle membrane / cellular response to lipopolysaccharide / presynaptic membrane / response to ethanol / G alpha (i) signalling events / perikaryon / phospholipase C-activating G protein-coupled receptor signaling pathway / defense response to virus / G alpha (q) signalling events / chemical synaptic transmission / dendritic spine / postsynaptic membrane / neuron projection / immune response / positive regulation of apoptotic process / G protein-coupled receptor signaling pathway / membrane raft / dendrite / positive regulation of gene expression / negative regulation of apoptotic process / protein-containing complex binding / cell surface / endoplasmic reticulum / Golgi apparatus / mitochondrion / nucleoplasm / identical protein binding / membrane / plasma membrane / cytosol
Similarity search - Function
Kappa opioid receptor / Neurotensin receptor / Neurotensin type 1 receptor / Opioid receptor / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
Chem-Q6Q / Neurotensin receptor type 1 / Kappa-type opioid receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsRobertson, M.J. / Skiniotis, G.
Funding support United States, 1items
OrganizationGrant numberCountry
The G. Harold and Leila Y. Mathers Foundation United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structure determination of inactive-state GPCRs with a universal nanobody.
Authors: Michael J Robertson / Makaía M Papasergi-Scott / Feng He / Alpay B Seven / Justin G Meyerowitz / Ouliana Panova / Maria Claudia Peroto / Tao Che / Georgios Skiniotis /
Abstract: 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 ...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.
History
DepositionApr 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 23, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Nov 30, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Dec 28, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Sep 25, 2024Group: Data collection / Source and taxonomy
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / em_entity_assembly_recombinant
Item: _em_admin.last_update / _em_entity_assembly_recombinant.id
Revision 1.5Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.6Nov 6, 2024Group: Data collection / Source and taxonomy / Category: em_admin / em_entity_assembly_naturalsource
Item: _em_admin.last_update / _em_entity_assembly_naturalsource.id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Neurotensin receptor 1
D: Nanobody 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4254
Polymers61,8152
Non-polymers6102
Water23413
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Neurotensin receptor 1 / NT-R-1 / NTR1 / High-affinity levocabastine-insensitive neurotensin receptor / NTRH / K-OR-1 / KOR-1


Mass: 47084.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NTSR1, NTRR, OPRK1, OPRK / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P30989, UniProt: P41145
#2: Antibody Nanobody 6


Mass: 14730.255 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-Q6Q / 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid


Mass: 587.065 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C32H31ClN4O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex of NTSR1 and Nb6COMPLEX#1-#20RECOMBINANT
2NTSR1COMPLEX#11RECOMBINANT
3Nb6COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Spodoptera frugiperda (fall armyworm)7108
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60.82 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 372987 / Symmetry type: POINT

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