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- PDB-7y13: Cryo-EM structure of apo-state MrgD-Gi complex (local) -

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Basic information

Entry
Database: PDB / ID: 7y13
TitleCryo-EM structure of apo-state MrgD-Gi complex (local)
ComponentsSoluble cytochrome b562,Mas-related G-protein coupled receptor member D
KeywordsSIGNALING PROTEIN / GPCR / Complex
Function / homology
Function and homology information


G protein-coupled receptor activity / electron transfer activity / periplasmic space / iron ion binding / G protein-coupled receptor signaling pathway / heme binding / extracellular space / plasma membrane
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-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
PALMITIC ACID / Soluble cytochrome b562 / Mas-related G-protein coupled receptor member D
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Homo sapiens (human)
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 1.1Nov 23, 2022Group: 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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)51,9368
Polymers50,1411
Non-polymers1,7957
Water362
1


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

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Components

#1: 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
#2: Chemical
ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C16H32O2
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
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

ComponentName: Soluble cytochrome b562,Mas-related G-protein coupled receptor member D
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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: 1.19.2_4158: / 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: 349331 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0032245
ELECTRON MICROSCOPYf_angle_d0.5943018
ELECTRON MICROSCOPYf_dihedral_angle_d5.643382
ELECTRON MICROSCOPYf_chiral_restr0.038351
ELECTRON MICROSCOPYf_plane_restr0.005350

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