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- EMDB-26589: CryoEM Structure of Inactive NTSR1 Bound to SR48692 and Nb6 -

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Basic information

Entry
Database: EMDB / ID: EMD-26589
TitleCryoEM Structure of Inactive NTSR1 Bound to SR48692 and Nb6
Map data
Sample
  • Complex: Complex of NTSR1 and Nb6
    • Complex: NTSR1Neurotensin receptor 1
      • Protein or peptide: Neurotensin receptor 1
    • Complex: Nb6
      • Protein or peptide: Nanobody 6Single-domain antibody
  • Ligand: 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid
  • Ligand: SODIUM IONSodium
  • Ligand: water
Function / homology
Function and homology information


dynorphin receptor activity / response to acrylamide / regulation of saliva secretion / negative regulation of luteinizing hormone secretion / positive regulation of eating behavior / sensory perception of temperature stimulus / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / adenylate cyclase-inhibiting opioid receptor signaling pathway / positive regulation of inhibitory postsynaptic potential ...dynorphin receptor activity / response to acrylamide / regulation of saliva secretion / negative regulation of luteinizing hormone secretion / positive regulation of eating behavior / sensory perception of temperature stimulus / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / adenylate cyclase-inhibiting opioid receptor signaling pathway / positive regulation of inhibitory postsynaptic potential / G protein-coupled opioid receptor activity / conditioned place preference / G protein-coupled opioid receptor signaling pathway / symmetric synapse / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / positive regulation of dopamine secretion / L-glutamate import across plasma membrane / positive regulation of potassium ion transmembrane transport / sensory perception / positive regulation of arachidonic acid secretion / maternal behavior / regulation of respiratory gaseous exchange / 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 inositol phosphate biosynthetic process / positive regulation of p38MAPK cascade / temperature homeostasis / eating behavior / response to lipid / regulation of membrane depolarization / behavioral response to cocaine / detection of temperature stimulus involved in sensory perception of pain / estrous cycle / neuropeptide signaling pathway / axon terminus / MECP2 regulates neuronal receptors and channels / sensory perception of pain / 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 / locomotory behavior / learning / G protein-coupled receptor activity / cellular response to glucose stimulus / response to insulin / terminal bouton / cytoplasmic side of plasma membrane / synaptic vesicle membrane / response to estrogen / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / chemical synaptic transmission / perikaryon / postsynaptic membrane / G alpha (q) signalling events / defense response to virus / response to ethanol / cellular response to lipopolysaccharide / dendritic spine / neuron projection / immune response / positive regulation of apoptotic process / G protein-coupled receptor signaling pathway / membrane raft / dendrite / protein-containing complex binding / positive regulation of gene expression / negative regulation of apoptotic process / Golgi apparatus / cell surface / endoplasmic reticulum / 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
Neurotensin receptor type 1 / Kappa-type opioid receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsRobertson MJ / Skiniotis G
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateDec 28, 2022-
Current statusDec 28, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26589.png

Downloads & links

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Map

FileDownload / File: emd_26589.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8677 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-4.67052 - 6.512369
Average (Standard dev.)-0.0009262758 (±0.0880241)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 277.664 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26589_additional_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_26589_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26589_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of NTSR1 and Nb6

EntireName: Complex of NTSR1 and Nb6
Components
  • Complex: Complex of NTSR1 and Nb6
    • Complex: NTSR1Neurotensin receptor 1
      • Protein or peptide: Neurotensin receptor 1
    • Complex: Nb6
      • Protein or peptide: Nanobody 6Single-domain antibody
  • Ligand: 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid
  • Ligand: SODIUM IONSodium
  • Ligand: water

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Supramolecule #1: Complex of NTSR1 and Nb6

SupramoleculeName: Complex of NTSR1 and Nb6 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: NTSR1

SupramoleculeName: NTSR1 / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nb6

SupramoleculeName: Nb6 / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Neurotensin receptor 1

MacromoleculeName: Neurotensin receptor 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.084391 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDAMG QPGNGSAFLL APNRSHAPDH DVENLYFQGQ RAQAGLEEAL LAPGFGNASG NASERVLAAP SSELDVNTDI YSKVLVTAV YLALFVVGTV GNTVTLFTLA RKKSLQSLQS TVHYHLGSLA LSDLLTLLLA MPVELYNFIW VHHPWAFGDA G CRGYYFLR ...String:
DYKDDDDAMG QPGNGSAFLL APNRSHAPDH DVENLYFQGQ RAQAGLEEAL LAPGFGNASG NASERVLAAP SSELDVNTDI YSKVLVTAV YLALFVVGTV GNTVTLFTLA RKKSLQSLQS TVHYHLGSLA LSDLLTLLLA MPVELYNFIW VHHPWAFGDA G CRGYYFLR DACTYATALN VASLSVERYL AICHPFKAKT LMSRSRTKKF ISAIWLASAL LAVPMLFTMG EQNRSADGQH AG GLVCTPT IHTATVKVVI QVNTFMSFIF PMVVISVLYT LMILRLKSVR LLSGSREKDR NLRRITRLVL AVVIAFVVCW LPY HVRRLM FCYISDEQWT PFLYDFYHYF YMVTNALFYV SSTINPILYN LVSANFRHIF LATLACLCPV WRRRRKRPAF SRKA DSVSS NHTLSSNATR ETLY

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Macromolecule #2: Nanobody 6

MacromoleculeName: Nanobody 6 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.730255 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQVQLQESG GGLVQAGESL RLSCAASGTI FRLYDMGWYR RVSGNQRELV ASITSGGSTK YGDSVKGRFT ISRDNAKNTV YLQMSSLKP EDTAVYYCNA EYRTGIWEEL LDGWGQGTQV TVSSHHHHHH EPEA

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Macromolecule #3: 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3...

MacromoleculeName: 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid
type: ligand / ID: 3 / Number of copies: 1 / Formula: Q6Q
Molecular weightTheoretical: 587.065 Da
Chemical component information

ChemComp-Q6Q:
2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 13 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.82 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 372987

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