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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: NTSR1
      • Protein or peptide: Neurotensin receptor 1
    • Complex: Nb6
      • Protein or peptide: Nanobody 6
  • Ligand: 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid
  • Ligand: SODIUM ION
  • Ligand: water
KeywordsAntagonist / Complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


response to acrylamide / regulation of saliva secretion / dynorphin receptor activity / sensory perception of temperature stimulus / positive regulation of eating behavior / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / adenylate cyclase-inhibiting opioid receptor signaling pathway / G protein-coupled opioid receptor activity / negative regulation of luteinizing hormone secretion ...response to acrylamide / regulation of saliva secretion / dynorphin receptor activity / sensory perception of temperature stimulus / positive regulation of eating behavior / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / adenylate cyclase-inhibiting opioid receptor signaling pathway / 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 / regulation of membrane depolarization / positive regulation of arachidonate secretion / L-glutamate import across plasma membrane / positive regulation of potassium ion transmembrane transport / regulation of respiratory gaseous exchange / positive regulation of inhibitory postsynaptic potential / maternal behavior / conditioned place preference / receptor serine/threonine kinase binding / neuropeptide 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 / detection of temperature stimulus involved in sensory perception of pain / behavioral response to cocaine / neuropeptide signaling pathway / estrous cycle / MECP2 regulates neuronal receptors and channels / axon terminus / 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 / sarcoplasmic reticulum / dendritic shaft / learning / locomotory behavior / G protein-coupled receptor activity / cellular response to glucose stimulus / response to nicotine / response to insulin / terminal bouton / synaptic vesicle membrane / cytoplasmic side of plasma membrane / response to estrogen / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / cellular response to lipopolysaccharide / chemical synaptic transmission / G alpha (q) signalling events / perikaryon / defense response to virus / postsynaptic membrane / response to ethanol / dendritic spine / neuron projection / immune response / positive regulation of apoptotic process / G protein-coupled receptor signaling pathway / membrane raft / dendrite / positive regulation of gene expression / protein-containing complex binding / negative regulation of apoptotic process / Golgi apparatus / cell surface / endoplasmic reticulum / 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
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-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing 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)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 320 pix.
= 277.664 Å		0.87 Å/pix.
x 320 pix.
= 277.664 Å		0.87 Å/pix.
x 320 pix.
= 277.664 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8677 Å
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Histogram

Histogram (log scale)
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
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Half map: #1

Fileemd_26589_half_map_1.map
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Half map: #2

Fileemd_26589_half_map_2.map
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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: NTSR1
      • Protein or peptide: Neurotensin receptor 1
    • Complex: Nb6
      • Protein or peptide: Nanobody 6
  • Ligand: 2-[[1-(7-chloranylquinolin-4-yl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl]carbonylamino]adamantane-2-carboxylic acid
  • Ligand: SODIUM ION
  • Ligand: water

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Supramolecule #1: Complex of NTSR1 and Nb6

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

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Supramolecule #3: Nb6

SupramoleculeName: Nb6 / type: complex / ID: 3 / 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

UniProtKB: Neurotensin receptor type 1, Kappa-type opioid receptor, Neurotensin receptor type 1

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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

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

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Image processing

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

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