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- PDB-9avg: Structure of human calcium-sensing receptor in complex with chime... -

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Basic information

Entry
Database: PDB / ID: 9avg
TitleStructure of human calcium-sensing receptor in complex with chimeric Gs (miniGis) protein in nanodiscs
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Chimeric mini guanine nucleotide-binding protein G(i)(s) subunit alpha
  • Isoform 1 of Extracellular calcium-sensing receptor
KeywordsMEMBRANE PROTEIN / Calcium-sensing receptor / G-protein-coupled receptor / G protein / signal transduction
Function / homology
Function and homology information


bile acid secretion / regulation of presynaptic membrane potential / chemosensory behavior / response to fibroblast growth factor / cellular response to peptide / cellular response to vitamin D / phosphatidylinositol-4,5-bisphosphate phospholipase C activity / Class C/3 (Metabotropic glutamate/pheromone receptors) / calcium ion import / positive regulation of positive chemotaxis ...bile acid secretion / regulation of presynaptic membrane potential / chemosensory behavior / response to fibroblast growth factor / cellular response to peptide / cellular response to vitamin D / phosphatidylinositol-4,5-bisphosphate phospholipase C activity / Class C/3 (Metabotropic glutamate/pheromone receptors) / calcium ion import / positive regulation of positive chemotaxis / fat pad development / cellular response to hepatocyte growth factor stimulus / amino acid binding / branching morphogenesis of an epithelial tube / positive regulation of calcium ion import / regulation of calcium ion transport / cellular response to low-density lipoprotein particle stimulus / anatomical structure morphogenesis / detection of calcium ion / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / T cell migration / D2 dopamine receptor binding / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / adenylate cyclase regulator activity / JNK cascade / positive regulation of vasoconstriction / axon terminus / regulation of mitotic spindle organization / cellular response to forskolin / chloride transmembrane transport / ossification / response to ischemia / Regulation of insulin secretion / positive regulation of cholesterol biosynthetic process / cellular response to glucose stimulus / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / G protein-coupled receptor binding / G protein-coupled receptor activity / positive regulation of insulin secretion / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / response to peptide hormone / 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 / G protein-coupled acetylcholine 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 / intracellular calcium ion homeostasis / 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 / vasodilation / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / cellular response to catecholamine stimulus / GDP binding / ADP signalling through P2Y purinoceptor 1 / 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 / integrin binding / cellular response to prostaglandin E stimulus / Inactivation, recovery and regulation of the phototransduction cascade / 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) / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G protein activity / presynaptic membrane / GTPase binding / Ca2+ pathway / retina development in camera-type eye / cell cortex / midbody / fibroblast proliferation / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events
Similarity search - Function
GPCR, family 3, extracellular calcium-sensing receptor-related / G-protein coupled receptors family 3 signature 1. / G-protein coupled receptors family 3 signature 2. / GPCR, family 3, nine cysteines domain / GPCR, family 3, nine cysteines domain superfamily / Nine Cysteines Domain of family 3 GPCR / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / G-protein coupled receptors family 3 profile. ...GPCR, family 3, extracellular calcium-sensing receptor-related / G-protein coupled receptors family 3 signature 1. / G-protein coupled receptors family 3 signature 2. / GPCR, family 3, nine cysteines domain / GPCR, family 3, nine cysteines domain superfamily / Nine Cysteines Domain of family 3 GPCR / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / G-protein coupled receptors family 3 profile. / GPCR family 3, C-terminal / 7 transmembrane sweet-taste receptor of 3 GCPR / G-protein alpha subunit, group S / G-protein alpha subunit, group I / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I / 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 beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / 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
Chem-9IG / : / PHOSPHATE ION / CYCLOMETHYLTRYPTOPHAN / CHOLESTEROL HEMISUCCINATE / Guanine nucleotide-binding protein G(s) subunit alpha / Extracellular calcium-sensing 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(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZuo, H. / Park, J. / Frangaj, A. / Ye, J. / Lu, G. / Manning, J.J. / Asher, W.B. / Lu, Z. / Hu, G. / Wang, L. ...Zuo, H. / Park, J. / Frangaj, A. / Ye, J. / Lu, G. / Manning, J.J. / Asher, W.B. / Lu, Z. / Hu, G. / Wang, L. / Mendez, J. / Eng, E. / Zhang, Z. / Lin, X. / Grasucci, R. / Hendrickson, W.A. / Clarke, O.B. / Javitch, J.A. / Conigrave, A.D. / Fan, Q.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141871 United States
CitationJournal: Nature / Year: 2024
Title: Promiscuous G-protein activation by the calcium-sensing receptor.
Authors: Hao Zuo / Jinseo Park / Aurel Frangaj / Jianxiang Ye / Guanqi Lu / Jamie J Manning / Wesley B Asher / Zhengyuan Lu / Guo-Bin Hu / Liguo Wang / Joshua Mendez / Edward Eng / Zhening Zhang / ...Authors: Hao Zuo / Jinseo Park / Aurel Frangaj / Jianxiang Ye / Guanqi Lu / Jamie J Manning / Wesley B Asher / Zhengyuan Lu / Guo-Bin Hu / Liguo Wang / Joshua Mendez / Edward Eng / Zhening Zhang / Xin Lin / Robert Grassucci / Wayne A Hendrickson / Oliver B Clarke / Jonathan A Javitch / Arthur D Conigrave / Qing R Fan /
Abstract: The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca ...The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca balance. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes. We determined structures of CaSR in complex with G proteins from three different subfamilies: G, G and G. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines G and G versus G selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.
History
DepositionMar 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 22, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Q: Isoform 1 of Extracellular calcium-sensing receptor
R: Isoform 1 of Extracellular calcium-sensing receptor
A: Chimeric mini guanine nucleotide-binding protein G(i)(s) subunit alpha
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,94428
Polymers280,4355
Non-polymers5,51023
Water00
1


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

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Components

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Protein , 2 types, 3 molecules QRA

#1: Protein Isoform 1 of Extracellular calcium-sensing receptor / CaR / CaSR / hCasR / Parathyroid cell calcium-sensing receptor 1 / PCaR1


Mass: 102864.617 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The CaSR construct consists of residues 1-903 and a Flag tag inserted after the signal peptide.
Source: (gene. exp.) Homo sapiens (human) / Gene: CASR, GPRC2A, PCAR1 / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: P41180
#2: Protein Chimeric mini guanine nucleotide-binding protein G(i)(s) subunit alpha / Adenylate cyclase-inhibiting G alpha protein


Mass: 28430.295 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Chimeric miniG(i)(s) alpha construct was designed where the N-terminal 25 amino acids of G(s) alpha was replaced with the initial 18 residues of G(i1) alpha
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1, GNAS / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: P63096, UniProt: A0A590UJY2

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

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


Mass: 38413.895 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 construct contains a Flag epitope fused to the N-terminus.
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: P62873
#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 / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: P59768

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Sugars , 2 types, 9 molecules

#5: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 6 types, 14 molecules

#7: Chemical ChemComp-TCR / CYCLOMETHYLTRYPTOPHAN


Type: L-peptide linking / Mass: 216.236 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C12H12N2O2
#8: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#9: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#10: Chemical ChemComp-9IG / 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine


Mass: 303.826 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H22ClNO
#11: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#12: Chemical ChemComp-A1AF7 / (19R,22S,25R)-22,25,26-trihydroxy-16,22-dioxo-17,21,23-trioxa-22lambda~5~-phosphahexacosan-19-yl (9Z)-octadec-9-enoate


Mass: 749.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H77O10P

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Details

Has ligand of interestN
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: Human CaSR in complex with chimeric Gs (miniGis) protein
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.276 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 GnTI-
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMSodium chlorideNaCl1
32 mMMagnesium chlorideMgCl21
410 mMCalcium chlorideCaCl21
520 uMTNCAC12H12N2O21
620 uMR568 HClC18H22ClNOHCl1
SpecimenConc.: 3.9 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 R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: The sample was blotted for 6s before plunge-frozen.

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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: 105000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K
Image recordingAverage exposure time: 2.72 sec. / Electron dose: 67.9 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 29091
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 15 eV
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2EPUimage acquisition
4cryoSPARC3.3.2CTF correction
7Coot0.9.8.1model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIX1.19.2_4158:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4867087
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45862 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
17SIL

7sil

Q7SILQ1
27SIL

7sil

R7SILR1
37P00

7p00

A7P00A2
47P00

7p00

B7P00B2
57P00

7p00

G7P00G2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00417907
ELECTRON MICROSCOPYf_angle_d0.62424328
ELECTRON MICROSCOPYf_dihedral_angle_d12.8886263
ELECTRON MICROSCOPYf_chiral_restr0.0432790
ELECTRON MICROSCOPYf_plane_restr0.0053057

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