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- PDB-8kgk: Cryo-EM structure of the GPR61-Gs complex -

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Basic information

Entry
Database: PDB / ID: 8kgk
TitleCryo-EM structure of the GPR61-Gs complex
Components
  • G-protein coupled receptor 61
  • 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(olf) subunit alpha,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Nanobody 35
KeywordsMEMBRANE PROTEIN / GPCR / Gs
Function / homology
Function and homology information


ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / Adenylate cyclase activating pathway / sensory perception of chemical stimulus / arrestin family protein binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress ...ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / Adenylate cyclase activating pathway / sensory perception of chemical stimulus / arrestin family protein binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / Hedgehog 'off' state / Adenylate cyclase inhibitory pathway / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / adenylate cyclase regulator activity / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / G protein-coupled receptor binding / G protein-coupled receptor activity / bone development / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / positive regulation of cold-induced thermogenesis / G protein activity / GTPase binding / Ca2+ pathway / retina development in camera-type eye / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / receptor complex / endosome / endosome membrane / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / metal ion binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
G-protein alpha subunit, group S / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily ...G-protein alpha subunit, group S / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / 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 / 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 / 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
Guanine nucleotide-binding protein G(olf) subunit alpha / 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 / G-protein coupled receptor 61
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsNie, Y. / Qiu, Z. / Zheng, S.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Nat Commun / Year: 2023
Title: Specific binding of GPR174 by endogenous lysophosphatidylserine leads to high constitutive G signaling.
Authors: Yingying Nie / Zeming Qiu / Sijia Chen / Zhao Chen / Xiaocui Song / Yan Ma / Niu Huang / Jason G Cyster / Sanduo Zheng /
Abstract: Many orphan G protein-coupled receptors (GPCRs) remain understudied because their endogenous ligands are unknown. Here, we show that a group of class A/rhodopsin-like orphan GPCRs including GPR61, ...Many orphan G protein-coupled receptors (GPCRs) remain understudied because their endogenous ligands are unknown. Here, we show that a group of class A/rhodopsin-like orphan GPCRs including GPR61, GPR161 and GPR174 increase the cAMP level similarly to fully activated D1 dopamine receptor (D1R). We report cryo-electron microscopy structures of the GPR61‒G, GPR161‒G and GPR174‒G complexes without any exogenous ligands. The GPR174 structure reveals that endogenous lysophosphatidylserine (lysoPS) is copurified. While GPR174 fails to respond to exogenous lysoPS, likely owing to its maximal activation by the endogenous ligand, GPR174 mutants with lower ligand binding affinities can be specifically activated by lysoPS but not other lipids, in a dose-dependent manner. Moreover, GPR174 adopts a non-canonical G coupling mode. The structures of GPR161 and GPR61 reveal that the second extracellular loop (ECL2) penetrates into the orthosteric pocket, possibly contributing to constitutive activity. Our work definitively confirms lysoPS as an endogenous GPR174 ligand and suggests that high constitutive activity of some orphan GPCRs could be accounted for by their having naturally abundant ligands.
History
DepositionAug 19, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: G-protein coupled receptor 61
B: Guanine nucleotide-binding protein G(olf) subunit alpha,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: Nanobody 35


Theoretical massNumber of molelcules
Total (without water)136,0215
Polymers136,0215
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 G-protein coupled receptor 61 / Biogenic amine receptor-like G-protein coupled receptor


Mass: 42527.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GPR61, BALGR, GPCR3 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9BZJ8
#2: Protein Guanine nucleotide-binding protein G(olf) subunit alpha,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein / olfactory type / Adenylate cyclase-stimulating G alpha protein


Mass: 29008.785 Da / Num. of mol.: 1
Mutation: G13R,V14N,D15E,E18A,R19Q,E33D,R34K,L35Q,A36V,K38R,G49D,E50N,A249D,S252D,I362A,V365I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAL, GNAS, GNAS1, GSP / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P38405, UniProt: P63092
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39286.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7845.078 Da / Num. of mol.: 1 / Mutation: C68S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768
#5: Antibody Nanobody 35


Mass: 17352.498 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)
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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of the GPR61 and mini-Gs complex with Nb35COMPLEXall0RECOMBINANT
2GPR61 and mini-GsCOMPLEX#1-#41RECOMBINANT
3Nb35COMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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 FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2480 nm / Nominal defocus min: 870 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 923

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Processing

EM softwareName: cryoSPARC / Version: V2 / Category: CTF correction / Details: patch CTF was used to determine the CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 277452
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192672 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027968
ELECTRON MICROSCOPYf_angle_d0.52910803
ELECTRON MICROSCOPYf_dihedral_angle_d4.2361098
ELECTRON MICROSCOPYf_chiral_restr0.0411228
ELECTRON MICROSCOPYf_plane_restr0.0041379

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