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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


Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Activation of the phototransduction cascade / Presynaptic function of Kainate receptors / G beta:gamma signalling through PLC beta / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through BTK / Glucagon-type ligand receptors / G alpha (z) signalling events ...Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Activation of the phototransduction cascade / Presynaptic function of Kainate receptors / G beta:gamma signalling through PLC beta / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through BTK / Glucagon-type ligand receptors / G alpha (z) signalling events / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / G alpha (s) signalling events / 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) / G-protein activation / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Activation of G protein gated Potassium channels / Ca2+ pathway / G alpha (q) signalling events / Extra-nuclear estrogen signaling / G alpha (12/13) signalling events / G alpha (i) signalling events / Vasopressin regulates renal water homeostasis via Aquaporins / GPER1 signaling / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / phototransduction, visible light / ADP signalling through P2Y purinoceptor 1 / alkylglycerophosphoethanolamine phosphodiesterase activity / photoreceptor outer segment membrane / spectrin binding / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / cell cortex region / G protein-coupled serotonin receptor binding / regulation of cAMP-mediated signaling / photoreceptor outer segment / cellular response to forskolin / regulation of mitotic spindle organization / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / G-protein beta/gamma-subunit complex binding / cardiac muscle cell apoptotic process / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / sensory perception of taste / Regulation of insulin secretion / photoreceptor inner segment / G-protein beta-subunit binding / GPER1 signaling / heterotrimeric G-protein complex / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G protein-coupled receptor activity / signaling receptor complex adaptor activity / response to peptide hormone / ADORA2B mediated anti-inflammatory cytokines production / phospholipase C-activating G protein-coupled receptor signaling pathway / GTPase binding / G protein-coupled receptor binding / retina development in camera-type eye / G alpha (s) signalling events / GDP binding / G alpha (i) signalling events / fibroblast proliferation / cell body / cell cortex / midbody / cellular response to hypoxia / cell population proliferation / myelin sheath / positive regulation of cytosolic calcium ion concentration / Extra-nuclear estrogen signaling / lysosomal membrane / electron transfer activity / periplasmic space / G protein-coupled receptor signaling pathway / cell cycle / GTPase activity / iron ion binding / cell division / centrosome / dendrite / protein-containing complex binding / GTP binding / heme binding / magnesium ion binding / extracellular space / extracellular exosome / membrane => GO:0016020 / plasma membrane / nucleus / cytoplasm
Similarity search - Function
Mas-related G protein-coupled receptor D / Mas-related G protein-coupled receptor family / Cytochrome b562 / Cytochrome b562 / G-protein alpha subunit, group I / Cytochrome c/b562 / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G protein alpha subunit, helical insertion ...Mas-related G protein-coupled receptor D / Mas-related G protein-coupled receptor family / Cytochrome b562 / Cytochrome b562 / G-protein alpha subunit, group I / Cytochrome c/b562 / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G protein alpha subunit, helical insertion / G protein alpha subunit / G-protein gamma subunit domain profile. / G-protein, gamma subunit / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / G-protein gamma-like domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / 7 transmembrane receptor (rhodopsin family) / GPCR, rhodopsin-like, 7TM / G protein-coupled receptor, rhodopsin-like / G-protein coupled receptors family 1 profile. / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats circular profile. / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / Trp-Asp (WD) repeats 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(i) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / BETA-ALANINE / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Soluble cytochrome b562 / PALMITIC ACID / 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 (unknown)
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: To Be Published
Title: Structural insight into the activation mechanism of MrgD with heterotrimeric Gi-protein
Authors: Suzuki, S. / Iida, M. / Hiroaki, Y. / Tanaka, K. / Kawamoto, A. / Kato, T. / Oshima, A.
History
DepositionJun 6, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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 (unknown) / 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 (unknown)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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