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- PDB-7wu9: Cryo-EM structure of the human EP3-Gi signaling complex -

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Basic information

Entry
Database: PDB / ID: 7wu9
TitleCryo-EM structure of the human EP3-Gi signaling complex
Components
  • 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
  • Prostaglandin E2 receptor EP3 subtype
  • scFv16
KeywordsSIGNALING PROTEIN / GPCR / signal transduction / MEMBRANE PROTEIN
Function / homology
Function and homology information


negative regulation of gastric acid secretion / intestine smooth muscle contraction / prostaglandin E receptor activity / Prostanoid ligand receptors / cell death / positive regulation of fever generation / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding ...negative regulation of gastric acid secretion / intestine smooth muscle contraction / prostaglandin E receptor activity / Prostanoid ligand receptors / cell death / positive regulation of fever generation / 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 / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Regulation of insulin secretion / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / 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 / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / response to peptide hormone / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / GDP binding / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / nuclear envelope / Ca2+ pathway / 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 / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / cell cycle / inflammatory response / G protein-coupled receptor signaling pathway / cell division / lysosomal membrane / GTPase activity / centrosome / synapse / protein-containing complex binding / GTP binding / nucleolus / magnesium ion binding / signal transduction / extracellular exosome / nucleoplasm / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Prostanoid EP3 receptor / Prostanoid EP3 receptor, type 2 / Prostaglandin DP receptor / Prostanoid receptor / 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 ...Prostanoid EP3 receptor / Prostanoid EP3 receptor, type 2 / Prostaglandin DP receptor / Prostanoid receptor / 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
Prostaglandin E2 receptor EP3 subtype / 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)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.375 Å
AuthorsSuno, R. / Sugita, Y. / Morimoto, K. / Iwasaki, K. / Kato, T. / Kobayashi, T.
Funding support Japan, 6items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21am0101072 (support number 1113), JP19am0101115 (support number 2365) Japan
Japan Agency for Medical Research and Development (AMED)JP21gm0910007 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0401020 Japan
Japan Society for the Promotion of Science (JSPS)15K08268, 19H03428,20H03434 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)21H05112 Japan
Japan Agency for Medical Research and Development (AMED)JP20ak010110 Japan
CitationJournal: Cell Rep / Year: 2022
Title: Structural insights into the G protein selectivity revealed by the human EP3-G signaling complex.
Authors: Ryoji Suno / Yukihiko Sugita / Kazushi Morimoto / Hiroko Takazaki / Hirokazu Tsujimoto / Mika Hirose / Chiyo Suno-Ikeda / Norimichi Nomura / Tomoya Hino / Asuka Inoue / Kenji Iwasaki / ...Authors: Ryoji Suno / Yukihiko Sugita / Kazushi Morimoto / Hiroko Takazaki / Hirokazu Tsujimoto / Mika Hirose / Chiyo Suno-Ikeda / Norimichi Nomura / Tomoya Hino / Asuka Inoue / Kenji Iwasaki / Takayuki Kato / So Iwata / Takuya Kobayashi /
Abstract: Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure ...Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-G signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of G to EP3 and the structural changes induced in EP3 by G binding. In addition, we compare the structure of the EP3-G complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to G that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.
History
DepositionFeb 7, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 1, 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
Revision 1.2Mar 8, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.3Oct 4, 2023Group: Data collection / Structure summary / Category: chem_comp_atom / chem_comp_bond / struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Prostaglandin E2 receptor EP3 subtype
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
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
S: scFv16


Theoretical massNumber of molelcules
Total (without water)149,5125
Polymers149,5125
Non-polymers00
Water0
1


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

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Components

#1: Protein Prostaglandin E2 receptor EP3 subtype / PGE receptor EP3 subtype / PGE2 receptor EP3 subtype / PGE2-R / Prostanoid EP3 receptor


Mass: 36175.887 Da / Num. of mol.: 1 / Mutation: A173I,V185S,N217Q,S258D,C289L,N308Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTGER3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P43115
#2: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40427.969 Da / Num. of mol.: 1
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
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37728.152 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: 7861.143 Da / Num. of mol.: 1
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 scFv16


Mass: 27318.389 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Brevibacillus agri (bacteria)

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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
1Prostaglandine E receptor EP3-Gi complexCOMPLEXall0MULTIPLE SOURCES
2Prostaglandine E receptor EP3-Gi complexCOMPLEX#1-#41RECOMBINANT
3scFv16COMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Brevibacillus agri (bacteria)51101
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 700 nm / Cs: 0.07 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 75 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.375 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125572 / Symmetry type: POINT

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