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- PDB-7bb7: AVP-V2R-Galphas-beta1-gamma2-Nb35(T state) -

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Basic information

Entry
Database: PDB / ID: 7bb7
TitleAVP-V2R-Galphas-beta1-gamma2-Nb35(T state)
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Nanobody 35Single-domain antibody
  • Vasopressin V2 receptorVasopressin receptor 2
  • Vasopressin
KeywordsMEMBRANE PROTEIN / Signaling protein / G protein-coupled receptor / Hormrne / Vasopressin / V2R
Function / homology
Function and homology information


renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / positive regulation of systemic arterial blood pressure / hemostasis / telencephalon development / positive regulation of intracellular signal transduction / PKA activation in glucagon signalling ...renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / positive regulation of systemic arterial blood pressure / hemostasis / telencephalon development / positive regulation of intracellular signal transduction / PKA activation in glucagon signalling / hair follicle placode formation / intracellular transport / endocytic vesicle / D1 dopamine receptor binding / developmental growth / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / positive regulation of vasoconstriction / cellular response to hormone stimulus / activation of adenylate cyclase activity / adenylate cyclase activator activity / trans-Golgi network membrane / response to cytokine / peptide binding / clathrin-coated endocytic vesicle membrane / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / bone development / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / platelet aggregation / Glucagon-type ligand receptors / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / sensory perception of smell / Thrombin signalling through proteinase activated receptors (PARs) / Cargo recognition for clathrin-mediated endocytosis / retina development in camera-type eye / GTPase binding / Clathrin-mediated endocytosis / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / endosome / G protein-coupled receptor signaling pathway / lysosomal membrane / negative regulation of cell population proliferation / GTPase activity / synapse / positive regulation of cell population proliferation / protein-containing complex binding / GTP binding / positive regulation of gene expression / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / signal transduction
Similarity search - Function
Vasopressin V2 receptor / Vasopressin receptor / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. ...Vasopressin V2 receptor / Vasopressin receptor / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / 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) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Vasopressin V2 receptor / 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(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsBous, J. / Mouillac, B. / Bron, P. / Granier, S. / Floquet, N. / Leyrat, C.
Funding support France, 2items
OrganizationGrant numberCountry
Foundation for Medical Research (France)DEQ20150331736 France
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0014 France
CitationJournal: Sci Adv / Year: 2021
Title: Cryo-electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex.
Authors: Julien Bous / Hélène Orcel / Nicolas Floquet / Cédric Leyrat / Joséphine Lai-Kee-Him / Gérald Gaibelet / Aurélie Ancelin / Julie Saint-Paul / Stefano Trapani / Maxime Louet / Rémy ...Authors: Julien Bous / Hélène Orcel / Nicolas Floquet / Cédric Leyrat / Joséphine Lai-Kee-Him / Gérald Gaibelet / Aurélie Ancelin / Julie Saint-Paul / Stefano Trapani / Maxime Louet / Rémy Sounier / Hélène Déméné / Sébastien Granier / Patrick Bron / Bernard Mouillac /
Abstract: The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the G protein, promoting kidney water reabsorption. Molecular mechanisms underlying ...The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the G protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-G complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and G binding modes. They reveal an original receptor-G interface in which the Gα subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.
History
DepositionDec 17, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 30, 2021Group: Structure summary / Category: audit_author

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Structure visualization

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Assembly

Deposited unit
A: Vasopressin V2 receptor
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
E: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
F: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
G: Nanobody 35
H: Vasopressin


Theoretical massNumber of molelcules
Total (without water)158,2076
Polymers158,2076
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15080 Å2
ΔGint-78 kcal/mol
Surface area45620 Å2
MethodPISA

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Components

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

#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 40507.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Plasmid: bacmid / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 45725.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Plasmid: bacmid / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63092
#4: 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 / Plasmid: bacmid / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Protein / Antibody / Protein/peptide , 3 types, 3 molecules AGH

#1: Protein Vasopressin V2 receptor / Vasopressin receptor 2 / V2R / AVPR V2 / Antidiuretic hormone receptor / Renal-type arginine vasopressin receptor


Mass: 47886.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AVPR2, ADHR, DIR, DIR3, V2R / Plasmid: bacmid / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P30518
#5: Antibody Nanobody 35 / Single-domain antibody


Mass: 15140.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria)
#6: Protein/peptide Vasopressin /


Mass: 1086.248 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: glycinamide c-terminal / Source: (synth.) Homo sapiens (human)

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Details

Has ligand of interestN

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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
1AVP-V2R-Galphas-beta1-gamma2-Nb35(T state)COMPLEXall0MULTIPLE SOURCES
2V2R and Guanine nucleotide-binding protein G(I)/G(S)/G(T)COMPLEX#1-#41RECOMBINANT
3Nanobody 35Single-domain antibodyCOMPLEX#51RECOMBINANT
4VasopressinCOMPLEX#61RECOMBINANT
Molecular weightValue: 0.156 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Lama glama (llama)9844
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli BL21(DE3) (bacteria)469008
34synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.UnitsFormulaBuffer-ID
1100 mMNaClSodium chloride1
220 mMHepes1
30.0011 %LMNG1
40.001 %GDN1
5%CHS1
610 microMAVP1
70.001 %A8-351
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1141.19COUNTINGGATAN K2 SUMMIT (4k x 4k)
2141.19COUNTINGGATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874+SVNrefinement
PHENIX1.18.2_3874+SVNrefinement
EM software
IDNameVersionCategory
1RELION3particle selection
4GctfCTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3566007
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 420953 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 72.14 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00688206
ELECTRON MICROSCOPYf_angle_d1.010311125
ELECTRON MICROSCOPYf_chiral_restr0.05561246
ELECTRON MICROSCOPYf_plane_restr0.00651430
ELECTRON MICROSCOPYf_dihedral_angle_d24.82821119

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