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- PDB-9m0d: Cryo-EM structure of neurotensin receptor 1 in complex with beta-... -

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Basic information

Entry
Database: PDB / ID: 9m0d
TitleCryo-EM structure of neurotensin receptor 1 in complex with beta-arrestin 1
Components
  • Beta-arrestin-1
  • Fab30 heavy chain
  • Fab30 light chain
  • Soluble cytochrome b562,Neurotensin receptor type 1
KeywordsMEMBRANE PROTEIN / GPCR / Neurotensin receptore 1 / Complex / beta-arrestin 1 / Phosphorylation
Function / homology
Function and homology information


G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / angiotensin receptor binding / symmetric synapse / D-aspartate import across plasma membrane / TGFBR3 regulates TGF-beta signaling / positive regulation of gamma-aminobutyric acid secretion / Activation of SMO / regulation of membrane depolarization / negative regulation of interleukin-8 production ...G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / angiotensin receptor binding / symmetric synapse / D-aspartate import across plasma membrane / TGFBR3 regulates TGF-beta signaling / positive regulation of gamma-aminobutyric acid secretion / Activation of SMO / regulation of membrane depolarization / negative regulation of interleukin-8 production / positive regulation of arachidonate secretion / L-glutamate import across plasma membrane / regulation of respiratory gaseous exchange / positive regulation of inhibitory postsynaptic potential / G protein-coupled receptor internalization / arrestin family protein binding / negative regulation of systemic arterial blood pressure / positive regulation of glutamate secretion / negative regulation of release of sequestered calcium ion into cytosol / Lysosome Vesicle Biogenesis / positive regulation of inositol phosphate biosynthetic process / response to lipid / temperature homeostasis / Golgi Associated Vesicle Biogenesis / positive regulation of Rho protein signal transduction / stress fiber assembly / positive regulation of cardiac muscle hypertrophy / negative regulation of NF-kappaB transcription factor activity / pseudopodium / negative regulation of interleukin-6 production / detection of temperature stimulus involved in sensory perception of pain / enzyme inhibitor activity / positive regulation of receptor internalization / negative regulation of Notch signaling pathway / neuropeptide signaling pathway / clathrin-coated pit / insulin-like growth factor receptor binding / negative regulation of protein ubiquitination / cytoplasmic vesicle membrane / GTPase activator activity / Activated NOTCH1 Transmits Signal to the Nucleus / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / Peptide ligand-binding receptors / adult locomotory behavior / learning / G protein-coupled receptor binding / electron transport chain / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / terminal bouton / G protein-coupled receptor activity / cytoplasmic side of plasma membrane / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / Signaling by BRAF and RAF1 fusions / endocytic vesicle membrane / protein transport / Cargo recognition for clathrin-mediated endocytosis / Thrombin signalling through proteinase activated receptors (PARs) / Clathrin-mediated endocytosis / positive regulation of protein phosphorylation / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / G alpha (s) signalling events / chemical synaptic transmission / molecular adaptor activity / G alpha (q) signalling events / dendritic spine / perikaryon / proteasome-mediated ubiquitin-dependent protein catabolic process / transcription coactivator activity / periplasmic space / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / Ub-specific processing proteases / protein ubiquitination / nuclear body / G protein-coupled receptor signaling pathway / positive regulation of apoptotic process / iron ion binding / membrane raft / Golgi membrane / lysosomal membrane / heme binding / ubiquitin protein ligase binding / positive regulation of gene expression / regulation of transcription by RNA polymerase II / protein-containing complex binding / negative regulation of apoptotic process / chromatin / cell surface / endoplasmic reticulum / Golgi apparatus / signal transduction / positive regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding
Similarity search - Function
Neurotensin receptor / Neurotensin type 1 receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain ...Neurotensin receptor / Neurotensin type 1 receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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) / Immunoglobulin E-set
Similarity search - Domain/homology
Soluble cytochrome b562 / Neurotensin receptor type 1 / Beta-arrestin-1
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsSun, D.M. / Li, X. / Yuan, Q.N. / Tian, C.L.
Funding support1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)T2221005
CitationJournal: Cell Res / Year: 2025
Title: Molecular mechanism of the arrestin-biased agonism of neurotensin receptor 1 by an intracellular allosteric modulator.
Authors: Demeng Sun / Xiang Li / Qingning Yuan / Yuanxia Wang / Pan Shi / Huanhuan Zhang / Tao Wang / Wenjing Sun / Shenglong Ling / Yuanchun Liu / Jinglin Lai / Wenqin Xie / Wanchao Yin / Lei Liu / ...Authors: Demeng Sun / Xiang Li / Qingning Yuan / Yuanxia Wang / Pan Shi / Huanhuan Zhang / Tao Wang / Wenjing Sun / Shenglong Ling / Yuanchun Liu / Jinglin Lai / Wenqin Xie / Wanchao Yin / Lei Liu / H Eric Xu / Changlin Tian /
Abstract: Biased allosteric modulators (BAMs) of G protein-coupled receptors (GPCRs) have been at the forefront of drug discovery owing to their potential to selectively stimulate therapeutically relevant ...Biased allosteric modulators (BAMs) of G protein-coupled receptors (GPCRs) have been at the forefront of drug discovery owing to their potential to selectively stimulate therapeutically relevant signaling and avoid on-target side effects. Although structures of GPCRs in complex with G protein or GRK in a BAM-bound state have recently been resolved, revealing that BAM can induce biased signaling by directly modulating interactions between GPCRs and these two transducers, no BAM-bound GPCR-arrestin complex structure has yet been determined, limiting our understanding of the full pharmacological profile of BAMs. Herein, we developed a chemical protein synthesis strategy to generate neurotensin receptor 1 (NTSR1) with defined hexa-phosphorylation at its C-terminus and resolved high-resolution cryo-EM structures (2.65-2.88 Å) of NTSR1 in complex with both β-arrestin1 and the BAM SBI-553. These structures revealed a unique "loop engagement" configuration of β-arrestin1 coupling to NTSR1 in the presence of SBI-553, markedly different from the typical "core engagement" configuration observed in the absence of BAMs. This configuration is characterized by the engagement of the intracellular loop 3 of NTSR1 with a cavity in the central crest of β-arrestin1, representing a previously unobserved, arrestin-selective conformation of GPCR. Our findings fill the critical knowledge gap regarding the regulation of GPCR-arrestin interactions and biased signaling by BAMs, which would advance the development of safer and more efficacious GPCR-targeted therapeutics.
History
DepositionFeb 24, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Soluble cytochrome b562,Neurotensin receptor type 1
B: Beta-arrestin-1
D: Fab30 heavy chain
Q: Fab30 light chain


Theoretical massNumber of molelcules
Total (without water)160,4724
Polymers160,4724
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Soluble cytochrome b562,Neurotensin receptor type 1 / Cytochrome b-562 / NT-R-1 / NTR1 / High-affinity levocabastine-insensitive neurotensin receptor / NTRH


Mass: 62097.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, NTSR1, NTRR
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
References: UniProt: P0ABE7, UniProt: P30989
#2: Protein Beta-arrestin-1 / Arrestin beta-1 / Non-visual arrestin-2


Mass: 45224.598 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARRB1, ARR1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P49407
#3: Antibody Fab30 heavy chain


Mass: 27727.010 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#4: Antibody Fab30 light chain


Mass: 25422.467 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
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

ComponentName: Ternary complex of neurotensin receptor 1 with beta-arrestin 1 and Fab30
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
Buffer solutionpH: 7.4
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6359592 / Symmetry type: POINT
RefinementHighest resolution: 3.41 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036504
ELECTRON MICROSCOPYf_angle_d0.5288975
ELECTRON MICROSCOPYf_dihedral_angle_d5.1731088
ELECTRON MICROSCOPYf_chiral_restr0.0411121
ELECTRON MICROSCOPYf_plane_restr0.0041205

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