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- EMDB-60056: Cryo-EM structure of the mmGPR4-Gs complex in pH7.6 -

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Entry
Database: EMDB / ID: EMD-60056
TitleCryo-EM structure of the mmGPR4-Gs complex in pH7.6
Map data
Sample
  • Complex: Cryo-EM structure of the mmGPR4-Gs complex in pH7.6
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: scFv16
    • Protein or peptide: G-protein coupled receptor 4
KeywordspH7.6 / mmGPR4 / Gs / MEMBRANE PROTEIN/IMMUNE SYSTEM / MEMBRANE PROTEIN / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Class A/1 (Rhodopsin-like receptors) / glomerular mesangial cell development / G alpha (q) signalling events / regulation of vascular permeability / response to acidic pH / angiogenesis involved in wound healing / positive regulation of Rho protein signal transduction / regulation of cell adhesion / negative regulation of angiogenesis / G protein-coupled receptor activity ...Class A/1 (Rhodopsin-like receptors) / glomerular mesangial cell development / G alpha (q) signalling events / regulation of vascular permeability / response to acidic pH / angiogenesis involved in wound healing / positive regulation of Rho protein signal transduction / regulation of cell adhesion / negative regulation of angiogenesis / G protein-coupled receptor activity / 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 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / positive regulation of inflammatory response / 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 / 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 / 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 / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / 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) / GTPase binding / Ca2+ pathway / retina development in camera-type eye / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / signal transduction / extracellular exosome / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
G protein-coupled receptor 4 orphan / 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-coupled receptor 4 orphan / 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 coupled receptors family 1 signature. / 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. / 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
Similarity search - Domain/homology
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 / G-protein coupled receptor 4
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsWen X / Rong NK / Yang F / Sun JP
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81773704 China
CitationJournal: Cell / Year: 2025
Title: Evolutionary study and structural basis of proton sensing by Mus GPR4 and Xenopus GPR4.
Authors: Xin Wen / Pan Shang / Haidi Chen / Lulu Guo / Naikang Rong / Xiaoyu Jiang / Xuan Li / Junyan Liu / Gongming Yang / Jiacheng Zhang / Kongkai Zhu / Qingbiao Meng / Xuefei He / Zhihai Wang / ...Authors: Xin Wen / Pan Shang / Haidi Chen / Lulu Guo / Naikang Rong / Xiaoyu Jiang / Xuan Li / Junyan Liu / Gongming Yang / Jiacheng Zhang / Kongkai Zhu / Qingbiao Meng / Xuefei He / Zhihai Wang / Zili Liu / Haoran Cheng / Yilin Zheng / Bifei Zhang / Jiaojiao Pang / Zhaoqian Liu / Peng Xiao / Yuguo Chen / Lunxu Liu / Fengming Luo / Xiao Yu / Fan Yi / Pengju Zhang / Fan Yang / Cheng Deng / Jin-Peng Sun /
Abstract: Animals have evolved pH-sensing membrane receptors, such as G-protein-coupled receptor 4 (GPR4), to monitor pH changes related to their physiology and generate adaptive reactions. However, the ...Animals have evolved pH-sensing membrane receptors, such as G-protein-coupled receptor 4 (GPR4), to monitor pH changes related to their physiology and generate adaptive reactions. However, the evolutionary trajectory and structural mechanism of proton sensing by GPR4 remain unresolved. Here, we observed a positive correlation between the optimal pH of GPR4 activity and the blood pH range across different species. By solving 7-cryoelectron microscopy (cryo-EM) structures of Xenopus tropicalis GPR4 (xtGPR4) and Mus musculus GPR4 (mmGPR4) under varying pH conditions, we identified that protonation of H and H enabled polar network establishment and tighter association between the extracellular loop 2 (ECL2) and 7 transmembrane (7TM) domain, as well as a conserved propagating path, which are common mechanisms underlying protonation-induced GPR4 activation across different species. Moreover, protonation of distinct extracellular H contributed to the more acidic optimal pH range of xtGPR4. Overall, our study revealed common and distinct mechanisms of proton sensing by GPR4, from a structural, functional, and evolutionary perspective.
History
DepositionMay 7, 2024-
Header (metadata) releaseFeb 26, 2025-
Map releaseFeb 26, 2025-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_60056.png

Downloads & links

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Map

FileDownload / File: emd_60056.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å		1.06 Å/pix.
x 300 pix.
= 318. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.007188065 - 1.9785215
Average (Standard dev.)0.0011777353 (±0.02520947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60056_msk_1.map
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Half map: #2

Fileemd_60056_half_map_1.map
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Half map: #1

Fileemd_60056_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of the mmGPR4-Gs complex in pH7.6

EntireName: Cryo-EM structure of the mmGPR4-Gs complex in pH7.6
Components
  • Complex: Cryo-EM structure of the mmGPR4-Gs complex in pH7.6
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: scFv16
    • Protein or peptide: G-protein coupled receptor 4

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Supramolecule #1: Cryo-EM structure of the mmGPR4-Gs complex in pH7.6

SupramoleculeName: Cryo-EM structure of the mmGPR4-Gs complex in pH7.6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.879465 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCTLSAEDK AAVERSKMIE KQLQKDKQVY RATHRLLLLG ADNSGKSTIV KQMRIYHVNG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI ...String:
MGCTLSAEDK AAVERSKMIE KQLQKDKQVY RATHRLLLLG ADNSGKSTIV KQMRIYHVNG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI PTQQDVLRTR VKTSGIFETK FQVDKVNFHM FDVGAQRDER RKWIQCFNDV TAIIFVVDSS DYNRLQEALN DF KSIWNNR WLRTISVILF LNKQDLLAEK VLAGKSKIED YFPEFARYTT PEDATPEPGE DPRVTRAKYF IRDEFLRIST ASG DGRHYC YPHFTCSVDT ENARRIFNDC RDIIQRMHLR QYELL

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.055867 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHGSL LQSELDQLRQ EAEQLKNQIR DARKACADAT LSQITNNIDP VGRIQMRTRR TLRGHLAKIY AMHWGTDSRL LVSASQDGK LIIWDSYTTN KVHAIPLRSS WVMTCAYAPS GNYVACGGLD NICSIYNLKT REGNVRVSRE LAGHTGYLSC C RFLDDNQI ...String:
MHHHHHHGSL LQSELDQLRQ EAEQLKNQIR DARKACADAT LSQITNNIDP VGRIQMRTRR TLRGHLAKIY AMHWGTDSRL LVSASQDGK LIIWDSYTTN KVHAIPLRSS WVMTCAYAPS GNYVACGGLD NICSIYNLKT REGNVRVSRE LAGHTGYLSC C RFLDDNQI VTSSGDTTCA LWDIETGQQT TTFTGHTGDV MSLSLAPDTR LFVSGACDAS AKLWDVREGM CRQTFTGHES DI NAICFFP NGNAFATGSD DATCRLFDLR ADQELMTYSH DNIICGITSV SFSKSGRLLL AGYDDFNCNV WDALKADRAG VLA GHDNRV SCLGVTDDGM AVATGSWDSF LKIWNGSSGG GGSGGGGSSG VSGWRLFKKI S

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.504446 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
NTASIAQARK LVEQLKMEAN IDRIKVSKAA ADLMAYCEAH AKEDPLLTPV PASENPFRE

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #4: scFv16

MacromoleculeName: scFv16 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 30.363043 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MLLVNQSHQG FNKEHTSKMV SAIVLYVLLA AAAHSAFAVQ LVESGGGLVQ PGGSRKLSCS ASGFAFSSFG MHWVRQAPEK GLEWVAYIS SGSGTIYYAD TVKGRFTISR DDPKNTLFLQ MTSLRSEDTA MYYCVRSIYY YGSSPFDFWG QGTTLTVSAG G GGSGGGGS ...String:
MLLVNQSHQG FNKEHTSKMV SAIVLYVLLA AAAHSAFAVQ LVESGGGLVQ PGGSRKLSCS ASGFAFSSFG MHWVRQAPEK GLEWVAYIS SGSGTIYYAD TVKGRFTISR DDPKNTLFLQ MTSLRSEDTA MYYCVRSIYY YGSSPFDFWG QGTTLTVSAG G GGSGGGGS GGGGSADIVM TQATSSVPVT PGESVSISCR SSKSLLHSNG NTYLYWFLQR PGQSPQLLIY RMSNLASGVP DR FSGSGSG TAFTLTISRL EAEDVGVYYC MQHLEYPLTF GAGTKLEL

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Macromolecule #5: G-protein coupled receptor 4

MacromoleculeName: G-protein coupled receptor 4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 41.149789 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDNSTGTGEG CHVDSRVDHL FPPSLYIFVI GVGLPTNCLA LWAAYRQVRQ HNELGVYLMN LSIADLLYIC TLPLWVDYFL HHDNWIHGP GSCKLFGFIF YSNIYISIAF LCCISVDRYL AVAHPLRFAR LRRVKTAVAV SSVVWATELG ANSAPLFHDE L FRDRYNHT ...String:
MDNSTGTGEG CHVDSRVDHL FPPSLYIFVI GVGLPTNCLA LWAAYRQVRQ HNELGVYLMN LSIADLLYIC TLPLWVDYFL HHDNWIHGP GSCKLFGFIF YSNIYISIAF LCCISVDRYL AVAHPLRFAR LRRVKTAVAV SSVVWATELG ANSAPLFHDE L FRDRYNHT FCFEKFPMER WVAWMNLYRV FVGFLFPWAL MLLCYRGILR AVQSSVSTER QEKVKIKRLA LSLIAIVLVC FA PYHALLL SRSAVYLGRP WDCGFEERVF SAYHSSLAFT SLNCVADPIL YCLVNEGARS DVAKALHNLL RFLASNKPQE MAN ASLTLE TPLTSKRSTT GKSSGAVWAV PPTAQGDQVP LKVLLPPAQ

UniProtKB: G-protein coupled receptor 4

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.875 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 490886
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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