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- PDB-9vba: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi... -

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Entry
Database: PDB / ID: 9vba
TitleCryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-closed C state 2, plunge-frozen 8 seconds after GTP addition
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • JMV449
  • Neurotensin receptor type 1
KeywordsMEMBRANE PROTEIN / GPCR / NTSR1 / neurotensin / G-protein
Function / homology
Function and homology information


G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / symmetric synapse / positive regulation of locomotion / regulation of inositol trisphosphate biosynthetic process / positive regulation of gamma-aminobutyric acid secretion / D-aspartate import across plasma membrane / positive regulation of arachidonate secretion / L-glutamate import across plasma membrane / vocalization behavior ...G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / symmetric synapse / positive regulation of locomotion / regulation of inositol trisphosphate biosynthetic process / positive regulation of gamma-aminobutyric acid secretion / D-aspartate import across plasma membrane / positive regulation of arachidonate secretion / L-glutamate import across plasma membrane / vocalization behavior / regulation of behavioral fear response / cAMP biosynthetic process / regulation of respiratory gaseous exchange / positive regulation of inhibitory postsynaptic potential / negative regulation of systemic arterial blood pressure / negative regulation of release of sequestered calcium ion into cytosol / positive regulation of glutamate secretion / response to food / regulation of membrane depolarization / response to lipid / temperature homeostasis / positive regulation of inositol phosphate biosynthetic process / detection of temperature stimulus involved in sensory perception of pain / response to stress / conditioned place preference / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / T cell migration / positive regulation of relaxation of smooth muscle / Adenylate cyclase inhibitory pathway / D2 dopamine receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / G protein-coupled serotonin receptor binding / cellular response to forskolin / Peptide ligand-binding receptors / positive regulation of release of sequestered calcium ion into cytosol / regulation of mitotic spindle organization / chemokine-mediated signaling pathway / dendritic shaft / adult locomotory behavior / Regulation of insulin secretion / neuropeptide signaling pathway / response to prostaglandin E / positive regulation of cholesterol biosynthetic process / negative regulation of insulin secretion / G protein-coupled receptor binding / response to peptide hormone / G protein-coupled receptor activity / cytoplasmic side of plasma membrane / centriolar satellite / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G protein-coupled acetylcholine receptor signaling pathway / terminal bouton / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Glucagon signaling in metabolic regulation / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G beta:gamma signalling through BTK / photoreceptor disc membrane / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / GDP binding / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / heterotrimeric G-protein complex / G alpha (12/13) signalling events / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / extracellular vesicle / sensory perception of taste / sperm principal piece / Thrombin signalling through proteinase activated receptors (PARs) / signaling receptor complex adaptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / retina development in camera-type eye / GTPase binding / fibroblast proliferation / G protein activity / midbody
Similarity search - Function
Neurotensin receptor / Neurotensin type 1 receptor / G-protein alpha subunit, group I / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / G-protein, gamma subunit / G-protein gamma subunit domain profile. ...Neurotensin receptor / Neurotensin type 1 receptor / G-protein alpha subunit, group I / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Neurotensin receptor type 1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 3.38 Å
AuthorsKobayashi, K. / Matsui, T.E. / Fukuda, M. / Kawakami, K. / Yamashita, K. / Kato, H.E.
Funding support Japan, 7items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)25K09525 Japan
Japan Society for the Promotion of Science (JSPS)24H02262 Japan
Japan Society for the Promotion of Science (JSPS)25H01338 Japan
Japan Society for the Promotion of Science (JSPS)24KJ0981 Japan
Japan Society for the Promotion of Science (JSPS)25H02243 Japan
Japan Society for the Promotion of Science (JSPS)24K18060 Japan
Japan Society for the Promotion of Science (JSPS)23KJ0363 Japan
CitationJournal: Nature / Year: 2026
Title: The dynamic basis of G-protein recognition and activation by a GPCR.
Authors: Kazuhiro Kobayashi / Kouki Kawakami / Toshiki E Matsui / Shun Yokoi / Masahiro Fukuda / Tomohiro J Narita / Hiroki Arai / Mai Tambo / Takashi Sumikama / Manae Tatsumi / Keitaro Yamashita / ...Authors: Kazuhiro Kobayashi / Kouki Kawakami / Toshiki E Matsui / Shun Yokoi / Masahiro Fukuda / Tomohiro J Narita / Hiroki Arai / Mai Tambo / Takashi Sumikama / Manae Tatsumi / Keitaro Yamashita / Junki Koyanagi / Mai Kugawa / Hisako Ikeda / Ayumi Sumino / Ayori Mitsutake / Brian K Kobilka / Asuka Inoue / Hideaki E Kato /
Abstract: G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes. Although more than 200 GPCR-G protein complex ...G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes. Although more than 200 GPCR-G protein complex structures have been determined, these static snapshots provide limited insight into the dynamics of G-protein association and dissociation. Here we present cryo-electron microscopy structures of human neurotensin receptor type 1 (NTSR1) with minimally modified G and G, showing how the receptor's intracellular surface dynamically rearranges to accommodate each G-protein subtype. Furthermore, time-resolved cryo-electron microscopy analyses of NTSR1-G visualized G-protein dissociation processes on GDP/GTP binding. Characterization of more than 20 intermediates, complemented by mutational and computational analyses, identifies four key mechanistic features. First, GDP/GTP induces G release from both canonical and non-canonical active conformations with distinct kinetics. Second, NTSR1 uses common intracellular rearrangements to recognize different G-protein subtypes and to promote activation of a single subtype. Third, separation from Gβγ involves stepwise remodelling of the Gα switches I-III. Finally, G dissociates from the receptor through a pathway that is distinct from that of G, and the canonical and non-canonical NTSR1-G complexes further diverge in their dissociation trajectories. These findings provide a comprehensive framework for understanding GPCR signalling dynamics and guiding signal-targeted therapeutic development.
History
DepositionJun 4, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
L: JMV449
R: Neurotensin receptor type 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,5156
Polymers136,9925
Non-polymers5231
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABC

#1: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40415.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39418.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein/peptide / Protein / Non-polymers , 3 types, 3 molecules LR

#4: Protein/peptide JMV449


Mass: 762.979 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Protein Neurotensin receptor type 1 / NT-R-1 / NTR1 / High-affinity levocabastine-insensitive neurotensin receptor / NTRH


Mass: 48535.051 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NTSR1, NTRR / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P30989
#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationN

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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
1JMV449-bound human Neurotensin Receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-closed C state 2 with scFv16, plunge-frozen 8 seconds after GTP additionCOMPLEX#1-#50MULTIPLE SOURCES
2JMV449-bound human neurotensin receptor type 1COMPLEX#1-#21RECOMBINANT
3GTP-bound heterotrimeric Gi1 proteinCOMPLEX#3-#41RECOMBINANT
4scFv16COMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181842 / Symmetry type: POINT
RefinementResolution: 3.38→3.38 Å / Cor.coef. Fo:Fc: 0.904 / SU B: 27.589 / SU ML: 0.441 / ESU R: 0.452
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.41606 --
obs0.41606 124292 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 158.819 Å2
Refinement stepCycle: 1 / Total: 9800
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0110.0129999
ELECTRON MICROSCOPYr_bond_other_d0.0040.0169185
ELECTRON MICROSCOPYr_angle_refined_deg2.3671.80313591
ELECTRON MICROSCOPYr_angle_other_deg1.3871.74221087
ELECTRON MICROSCOPYr_dihedral_angle_1_deg9.64251273
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.558259
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.07101584
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.1390.21568
ELECTRON MICROSCOPYr_gen_planes_refined0.0130.0211876
ELECTRON MICROSCOPYr_gen_planes_other0.0080.022362
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it8.31815.7975115
ELECTRON MICROSCOPYr_mcbond_other8.31715.7985115
ELECTRON MICROSCOPYr_mcangle_it14.23728.6256379
ELECTRON MICROSCOPYr_mcangle_other14.23628.6266380
ELECTRON MICROSCOPYr_scbond_it8.22616.4684884
ELECTRON MICROSCOPYr_scbond_other8.22616.4684884
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other14.84430.2277213
ELECTRON MICROSCOPYr_long_range_B_refined28.954188.3539473
ELECTRON MICROSCOPYr_long_range_B_other28.954188.3439474
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.2→3.283 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.162 9165 -
obs--100 %

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