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- PDB-7y12: Cryo-EM structure of MrgD-Gi complex with beta-alanine -

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Basic information

Entry
Database: PDB / ID: 7y12
TitleCryo-EM structure of MrgD-Gi complex with beta-alanine
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Soluble cytochrome b562,Mas-related G-protein coupled receptor member D
  • scFV16
KeywordsSIGNALING PROTEIN / GPCR / Complex
Function / homology
Function and homology information


Activation of the phototransduction cascade / Sensory perception of sweet, bitter, and umami (glutamate) taste / Olfactory Signaling Pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 ...Activation of the phototransduction cascade / Sensory perception of sweet, bitter, and umami (glutamate) taste / Olfactory Signaling Pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G beta:gamma signalling through BTK / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thromboxane signalling through TP receptor / Thrombin signalling through proteinase activated receptors (PARs) / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Ca2+ pathway / G alpha (s) signalling events / G alpha (q) signalling events / Extra-nuclear estrogen signaling / G alpha (12/13) signalling events / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (i) signalling events / GPER1 signaling / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADP signalling through P2Y purinoceptor 1 / phototransduction, visible light / alkylglycerophosphoethanolamine phosphodiesterase activity / photoreceptor outer segment membrane / spectrin binding / photoreceptor outer segment / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / cardiac muscle cell apoptotic process / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / photoreceptor inner segment / Regulation of insulin secretion / G protein-coupled receptor binding / G protein-coupled receptor activity / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / response to peptide hormone / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / GPER1 signaling / GDP binding / G-protein beta-subunit binding / heterotrimeric G-protein complex / signaling receptor complex adaptor activity / retina development in camera-type eye / GTPase binding / myelin sheath / cell body / phospholipase C-activating G protein-coupled receptor signaling pathway / cell cortex / midbody / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / fibroblast proliferation / cellular response to hypoxia / G alpha (s) signalling events / cell population proliferation / Extra-nuclear estrogen signaling / electron transfer activity / periplasmic space / cell cycle / iron ion binding / G protein-coupled receptor signaling pathway / cell division / lysosomal membrane / GTPase activity / centrosome / dendrite / heme binding / protein-containing complex binding / GTP binding / nucleolus / magnesium ion binding / extracellular space / extracellular exosome / nucleoplasm / plasma membrane / cytoplasm
Similarity search - Function
Mas-related G protein-coupled receptor D / Mas-related G protein-coupled receptor family / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein alpha subunit, group I / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit ...Mas-related G protein-coupled receptor D / Mas-related G protein-coupled receptor family / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein alpha subunit, group I / 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 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
BETA-ALANINE / PALMITIC ACID / Soluble cytochrome b562 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Mas-related G-protein coupled receptor member D
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
Escherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSuzuki, S. / Iida, M. / Kawamoto, A. / Oshima, A.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19H03165 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101074 Japan
CitationJournal: Commun Biol / Year: 2022
Title: Structural insight into the activation mechanism of MrgD with heterotrimeric Gi-protein revealed by cryo-EM.
Authors: Shota Suzuki / Momoko Iida / Yoko Hiroaki / Kotaro Tanaka / Akihiro Kawamoto / Takayuki Kato / Atsunori Oshima /
Abstract: MrgD, a member of the Mas-related G protein-coupled receptor (MRGPR) family, has high basal activity for Gi activation. It recognizes endogenous ligands, such as β-alanine, and is involved in pain ...MrgD, a member of the Mas-related G protein-coupled receptor (MRGPR) family, has high basal activity for Gi activation. It recognizes endogenous ligands, such as β-alanine, and is involved in pain and itch signaling. The lack of a high-resolution structure for MrgD hinders our understanding of whether its activation is ligand-dependent or constitutive. Here, we report two cryo-EM structures of the MrgD-Gi complex in the β-alanine-bound and apo states at 3.1 Å and 2.8 Å resolution, respectively. These structures show that β-alanine is bound to a shallow pocket at the extracellular domains. The extracellular half of the sixth transmembrane helix undergoes a significant movement and is tightly packed into the third transmembrane helix through hydrophobic residues, creating the active form. Our structures demonstrate a structural basis for the characteristic ligand recognition of MrgD. These findings provide a framework to guide drug designs targeting the MrgD receptor.
History
DepositionJun 6, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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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
R: Soluble cytochrome b562,Mas-related G-protein coupled receptor member D
S: scFV16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,5079
Polymers169,6495
Non-polymers8584
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 40446.047 Da / Num. of mol.: 1 / Mutation: G203A, T219A, P288Q, A326S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39461.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gnb1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62874
#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.) Mus musculus (house mouse) / Gene: Gng2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63213

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Protein / Antibody , 2 types, 2 molecules RS

#4: Protein Soluble cytochrome b562,Mas-related G-protein coupled receptor member D / Cytochrome b-562 / Beta-alanine receptor / G-protein coupled receptor TGR7


Mass: 50141.285 Da / Num. of mol.: 1
Fragment: Chimera protein of Cytochrome b-562 (UNP residues 23-127) and MrgD (UNP residues 5-321)
Mutation: M29W, H124I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, MRGPRD, MRGD / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0ABE7, UniProt: Q8TDS7
#5: Antibody scFV16


Mass: 31739.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Spodoptera frugiperda (fall armyworm)

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Non-polymers , 2 types, 4 molecules

#6: Chemical ChemComp-BAL / BETA-ALANINE / Β-Alanine


Type: peptide-like / Mass: 89.093 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7NO2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C16H32O2

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Details

Has ligand of interestY

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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
1beta-alanine bound MrgD-Gi complex with the scFv16COMPLEX#1-#50RECOMBINANT
2G(i) subunit alpha-1,Mas-related G-protein coupled receptor member DCOMPLEX#1, #41RECOMBINANT
3subunit beta-1,subunit gamma-2COMPLEX#2-#31RECOMBINANT
4scFV16COMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Mus musculus (house mouse)10090
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
43Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.4
SpecimenConc.: 10 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: 277 K / Details: blot time 3 seconds blot force 5

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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 / Nominal defocus max: 1900 nm / Nominal defocus min: 700 nm / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsSpherical aberration corrector: The Microscope implicated Cs corrector.

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
8UCSF ChimeraXmodel fitting
10PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97282 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038683
ELECTRON MICROSCOPYf_angle_d0.5111767
ELECTRON MICROSCOPYf_dihedral_angle_d4.9261238
ELECTRON MICROSCOPYf_chiral_restr0.041359
ELECTRON MICROSCOPYf_plane_restr0.0031469

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