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- EMDB-61104: cryo-EM structure of zebrafish GPR4-Gs complex at pH 8.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-61104
Titlecryo-EM structure of zebrafish GPR4-Gs complex at pH 8.5
Map data
Sample
  • Complex: zGPR4-Gs complex
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: scfv16
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Soluble cytochrome b562,G-protein coupled receptor 4,lgBit
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
KeywordsGPCR / proton-sensing / G protein / SIGNALING PROTEIN/IMMUNE SYSTEM / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


response to acidic pH / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway ...response to acidic pH / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / electron transport chain / G protein-coupled receptor activity / bone development / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / 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 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / 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 / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / 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) / positive regulation of cold-induced thermogenesis / G protein activity / 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 / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / periplasmic space / electron transfer activity / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway / iron ion binding / lysosomal membrane / GTPase activity / synapse / heme binding / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / metal ion binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
G protein-coupled receptor 4 orphan / G-protein alpha subunit, group S / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit ...G protein-coupled receptor 4 orphan / G-protein alpha subunit, group S / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / 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. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular 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
G-protein coupled receptor 4 / Soluble cytochrome b562 / 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(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesDanio rerio (zebrafish) / Homo sapiens (human) / Mus musculus (house mouse) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsMa YT / Tang MY / Song GJ / Ru H
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32171215 China
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM structure of an activated GPR4-Gs signaling complex.
Authors: Yitong Ma / Yijie Wang / Mengyuan Tang / Yuan Weng / Ying Chen / Yueming Xu / Shuxiao An / Yiran Wu / Suwen Zhao / Huanhuan Xu / Dali Li / Mingyao Liu / Weiqiang Lu / Heng Ru / Gaojie Song /
Abstract: G protein-coupled receptor 4 (GPR4) belongs to the subfamily of proton-sensing GPCRs (psGPCRs), which detect pH changes in extracellular environment and regulate diverse physiological responses. GPR4 ...G protein-coupled receptor 4 (GPR4) belongs to the subfamily of proton-sensing GPCRs (psGPCRs), which detect pH changes in extracellular environment and regulate diverse physiological responses. GPR4 was found to be overactivated in acidic tumor microenvironment as well as inflammation sites, with a triad of acidic residues within the transmembrane domain identified as crucial for proton sensing. However, the 3D structure remains unknown, and the roles of other conserved residues within psGPCRs are not well understood. Here we report cryo-electron microscopy (cryo-EM) structures of active zebrafish GPR4 at both pH 6.5 and 8.5, each highlighting a distribution of histidine and acidic residues at the extracellular region. Cell-based assays show that these ionizable residues moderately influence the proton-sensing capacity of zebrafish GPR4, compared to the more significant effects of the triad residues. Furthermore, we reveal a cluster of aromatic residues within the orthosteric pocket that may propagate the signaling to the intercellular region via repacking the aromatic patch at the central region. This study provides a framework for future signaling and functional investigation of psGPCRs.
History
DepositionAug 7, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
SupersessionFeb 19, 2025ID: EMD-60062
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61104.png

Downloads & links

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Map

FileDownload / File: emd_61104.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.394
Minimum - Maximum-2.8820958 - 4.2771764
Average (Standard dev.)-0.0007696892 (±0.08991678)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 238.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61104_msk_1.map
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Additional map: The additional cryo-EM map was refined from further...

Fileemd_61104_additional_1.map
AnnotationThe additional cryo-EM map was refined from further 3D classification of the main map, which showed better local resolution at the receptor region and improved the model building.
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_61104_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_61104_half_map_2.map
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Sample components

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Entire : zGPR4-Gs complex

EntireName: zGPR4-Gs complex
Components
  • Complex: zGPR4-Gs complex
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: scfv16
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Soluble cytochrome b562,G-protein coupled receptor 4,lgBit
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Supramolecule #1: zGPR4-Gs complex

SupramoleculeName: zGPR4-Gs complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Danio rerio (zebrafish)

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Macromolecule #1: 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: 1
Details: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, with C-terminal smBIT tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.728426 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL ...String:
MSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQTFTGHESD INAICFFPNG NA FATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DALKADRAGV LAGHDNRVSC LGV TDDGMA VATGSWDSFL KIWNGSSGGG GSGGGGSSGV SGWRLFKKIS

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

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Macromolecule #2: scfv16

MacromoleculeName: scfv16 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 31.560316 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MLLVNQSHQG FNKEHTSKMV SAIVLYVLLA AAAHSAFADV QLVESGGGLV QPGGSRKLSC SASGFAFSSF GMHWVRQAPE KGLEWVAYI SSGSGTIYYA DTVKGRFTIS RDDPKNTLFL QMTSLRSEDT AMYYCVRSIY YYGSSPFDFW GQGTTLTVSS G GGGSGGGG ...String:
MLLVNQSHQG FNKEHTSKMV SAIVLYVLLA AAAHSAFADV QLVESGGGLV QPGGSRKLSC SASGFAFSSF GMHWVRQAPE KGLEWVAYI SSGSGTIYYA DTVKGRFTIS RDDPKNTLFL QMTSLRSEDT AMYYCVRSIY YYGSSPFDFW GQGTTLTVSS G GGGSGGGG SGGGGSDIVM TQATSSVPVT PGESVSISCR SSKSLLHSNG NTYLYWFLQR PGQSPQLLIY RMSNLASGVP DR FSGSGSG TAFTLTISRL EAEDVGVYYC MQHLEYPLTF GAGTKLELKA AAENLYFQ

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Macromolecule #3: 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: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.362049 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GSATVSAEDK AAAERSKMIE KQLQKDKQVY RATHRLLLLG ADNSGKSTIV KQMRIYHGGS GGSGGTSGIF ETKFQVDKVN FHMFDVGGQ RDERRKWIQC FNDVTAIIFV VDSSDYNRLQ EALNDFKSIW NNRWLRTISV ILFLNKQDLL AEKVLAGKSK I EDYFPEFA ...String:
GSATVSAEDK AAAERSKMIE KQLQKDKQVY RATHRLLLLG ADNSGKSTIV KQMRIYHGGS GGSGGTSGIF ETKFQVDKVN FHMFDVGGQ RDERRKWIQC FNDVTAIIFV VDSSDYNRLQ EALNDFKSIW NNRWLRTISV ILFLNKQDLL AEKVLAGKSK I EDYFPEFA RYTTPEDATP EPGEDPRVTR AKYFIRDEFL RISTASGDGR HYCYPHFTCA VDTENARRIF NDCRDIIQRM HL RQYELL

UniProtKB: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Macromolecule #4: Soluble cytochrome b562,G-protein coupled receptor 4,lgBit

MacromoleculeName: Soluble cytochrome b562,G-protein coupled receptor 4,lgBit
type: protein_or_peptide / ID: 4
Details: GPR4 with N-terminal BRIL (from E. coli., density missed) protein and C-terminal lgBit tag (synthetic peptide, density missed)
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 72.372461 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAADLEDN WETLNDNLKV IEKADNAAQV KDALTKMRAA ALDAQKATPP KLEDKSPDSP EMKDFRHGF DILVGQIDDA LKLANEGKVK EAQAAAEQLK TTRNAYIQKY LASLRGSETM CNITTSCNVD SNIDQFFQPT L YIIVIVLG ...String:
MKTIIALSYI FCLVFADYKD DDDAADLEDN WETLNDNLKV IEKADNAAQV KDALTKMRAA ALDAQKATPP KLEDKSPDSP EMKDFRHGF DILVGQIDDA LKLANEGKVK EAQAAAEQLK TTRNAYIQKY LASLRGSETM CNITTSCNVD SNIDQFFQPT L YIIVIVLG FPTNCMALWA AYMQVKQKNE LGIYLMNLSI ADLLYITTLP LWIDYFVHHD NWIHGQVSCK LFGFIFYTNI YV SIAFLCC ISVDRYLAVA HPLKFAKVRR VKTALLVSAI VWLTEIVANS APLFHDELFQ DRFNHTFCFE KYPMEPWVAG MNL YRTFLG FLAPWGIMLA AYRGILRAVR GNVSTERQEK AKIKRLALSL ILIVLLCFAP YHVLLLWRSV LFLINPCDCG GEEN LFGAY HVTLALTSLN CVADPILYCF VNEGARHDVG RALATLLGLF QRGKSPETLM GASITVETPL AVVFTLEDFV GDWEQ TAAY NLDQVLEQGG VSSLLQNLAV SVTPIQRIVR SGENALKIDI HVIIPYEGLS ADQMAQIEEV FKVVYPVDDH HFKVIL PYG TLVIDGVTPN MLNYFGRPYE GIAVFDGKKI TVTGTLWNGN KIIDERLITP DGSMLFRVTI NLEVLFQGPH HHHHHHH HH

UniProtKB: Soluble cytochrome b562, G-protein coupled receptor 4

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Macromolecule #5: 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: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.675814 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFGSA

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

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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

Concentration12 mg/mL
BufferpH: 8.5
Details: 20 mM MES (pH 6.5), 100 mM NaCl, 2 mM MgCl2, 0.0025% (w/v) LMNG, and 0.0005% (w/v) CHS.
VitrificationCryogen name: ETHANE
Detailsthis complex was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 314824
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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