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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


dynorphin receptor activity / response to acrylamide / regulation of saliva secretion / sensory perception of temperature stimulus / positive regulation of eating behavior / G protein-coupled neurotensin receptor activity / adenylate cyclase-inhibiting opioid receptor signaling pathway / inositol phosphate catabolic process / G protein-coupled opioid receptor activity / negative regulation of luteinizing hormone secretion ...dynorphin receptor activity / response to acrylamide / regulation of saliva secretion / sensory perception of temperature stimulus / positive regulation of eating behavior / G protein-coupled neurotensin receptor activity / adenylate cyclase-inhibiting opioid receptor signaling pathway / inositol phosphate catabolic process / G protein-coupled opioid receptor activity / negative regulation of luteinizing hormone secretion / symmetric synapse / G protein-coupled opioid receptor signaling pathway / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / positive regulation of dopamine secretion / sensory perception / L-glutamate import across plasma membrane / positive regulation of potassium ion transmembrane transport / positive regulation of arachidonic acid secretion / conditioned place preference / regulation of respiratory gaseous exchange / positive regulation of inhibitory postsynaptic potential / maternal behavior / neuropeptide binding / receptor serine/threonine kinase binding / negative regulation of systemic arterial blood pressure / negative regulation of release of sequestered calcium ion into cytosol / positive regulation of glutamate secretion / positive regulation of p38MAPK cascade / positive regulation of inositol phosphate biosynthetic process / temperature homeostasis / eating behavior / response to lipid / regulation of membrane depolarization / detection of temperature stimulus involved in sensory perception of pain / behavioral response to cocaine / estrous cycle / neuropeptide signaling pathway / axon terminus / MECP2 regulates neuronal receptors and channels / sensory perception of pain / T-tubule / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Peptide ligand-binding receptors / adult locomotory behavior / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / learning / sarcoplasmic reticulum / locomotory behavior / G protein-coupled receptor activity / cellular response to glucose stimulus / response to nicotine / response to insulin / terminal bouton / cytoplasmic side of plasma membrane / synaptic vesicle membrane / response to estrogen / phospholipase C-activating G protein-coupled receptor signaling pathway / presynaptic membrane / G alpha (i) signalling events / G alpha (q) signalling events / chemical synaptic transmission / postsynaptic membrane / perikaryon / response to ethanol / defense response to virus / cellular response to lipopolysaccharide / dendritic spine / neuron projection / immune response / membrane raft / positive regulation of apoptotic process / G protein-coupled receptor signaling pathway / dendrite / protein-containing complex binding / positive regulation of gene expression / negative regulation of apoptotic process / Golgi apparatus / cell surface / endoplasmic reticulum / mitochondrion / nucleoplasm / membrane / identical protein binding / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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

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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
21Homo sapiens (human)9606
32Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera 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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