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- EMDB-61439: Cryo-EM structure of GPR65 complexed with miniGs in pH6.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-61439
TitleCryo-EM structure of GPR65 complexed with miniGs in pH6.5
Map data
Sample
  • Complex: gpr65-gs
    • Complex: GPCR-GS
      • 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: Psychosine receptor
    • Complex: nanobody
      • Protein or peptide: Nanobody 35
KeywordsGPCR / GPR65 / miniGs / Proton sensing / SIGNALING PROTEIN / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


response to acidic pH / Class A/1 (Rhodopsin-like receptors) / positive regulation of stress fiber assembly / 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 ...response to acidic pH / Class A/1 (Rhodopsin-like receptors) / positive regulation of stress fiber assembly / 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 / 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 / ADP signalling through P2Y purinoceptor 1 / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / 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 / 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) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / fibroblast proliferation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / immune response / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / apoptotic process / synapse / protein-containing complex binding / signal transduction / extracellular exosome / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Psychosine receptor / 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 / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit ...Psychosine receptor / 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 / G protein beta WD-40 repeat protein / 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. / 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 65
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsYue XL / Wu LJ / Hua T / Liu ZJ
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Res / Year: 2025
Title: Structural basis of stepwise proton sensing-mediated GPCR activation.
Authors: Xiaolei Yue / Li Peng / Shenhui Liu / Bingjie Zhang / Xiaodan Zhang / Hao Chang / Yuan Pei / Xiaoting Li / Junlin Liu / Wenqing Shui / Lijie Wu / Huji Xu / Zhi-Jie Liu / Tian Hua /
Abstract: The regulation of pH homeostasis is crucial in many biological processes vital for survival, growth, and function of life. The pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 ...The regulation of pH homeostasis is crucial in many biological processes vital for survival, growth, and function of life. The pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 and GPR68, play a pivotal role in detecting changes in extracellular proton concentrations, impacting both physiological and pathological states. However, comprehensive understanding of the proton sensing mechanism is still elusive. Here, we determined the cryo-electron microscopy structures of GPR4 and GPR65 in various activation states across different pH levels, coupled with G, G or G proteins, as well as a small molecule NE52-QQ57-bound inactive GPR4 structure. These structures reveal the dynamic nature of the extracellular loop 2 and its signature conformations in different receptor states, and disclose the proton sensing mechanism mediated by networks of extracellular histidine and carboxylic acid residues. Notably, we unexpectedly captured partially active intermediate states of both GPR4-G and GPR4-G complexes, and identified a unique allosteric binding site for NE52-QQ57 in GPR4. By integrating prior investigations with our structural analysis and mutagenesis data, we propose a detailed atomic model for stepwise proton sensation and GPCR activation. These insights may pave the way for the development of selective ligands and targeted therapeutic interventions for pH sensing-relevant diseases.
History
DepositionSep 5, 2024-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateAug 13, 2025-
Current statusAug 13, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61439.png

Downloads & links

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Map

FileDownload / File: emd_61439.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.00091648893 - 2.005683
Average (Standard dev.)0.001874486 (±0.034250543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_61439_half_map_1.map
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Half map: #1

Fileemd_61439_half_map_2.map
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Sample components

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Entire : gpr65-gs

EntireName: gpr65-gs
Components
  • Complex: gpr65-gs
    • Complex: GPCR-GS
      • 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: Psychosine receptor
    • Complex: nanobody
      • Protein or peptide: Nanobody 35

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Supramolecule #1: gpr65-gs

SupramoleculeName: gpr65-gs / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: GPCR-GS

SupramoleculeName: GPCR-GS / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3, #5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: nanobody

SupramoleculeName: nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Lama glama (llama)

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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: 29.038883 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNSKTEDQRN EEKAQREANK KIEKQLQKDK QVYRATHRLL LLGADNSGKS TIVKQMRILH GGSGGSGGTS GIFETKFQVD KVNFHMFDV GGQRDERRKW IQCFNDVTAI IFVVDSSDYN RLQEALNLFK SIWNNRWLRT ISVILFLNKQ DLLAEKVLAG K SKIEDYFP ...String:
MNSKTEDQRN EEKAQREANK KIEKQLQKDK QVYRATHRLL LLGADNSGKS TIVKQMRILH GGSGGSGGTS GIFETKFQVD KVNFHMFDV GGQRDERRKW IQCFNDVTAI IFVVDSSDYN RLQEALNLFK SIWNNRWLRT ISVILFLNKQ DLLAEKVLAG K SKIEDYFP EFARYTTPED ATPEPGEDPR VTRAKYFIRD EFLRISTASG DGRHYCYPHF TCAVDTENAR RIFNDCRDII QR MHLRQYE LL

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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: 37.41693 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 KIWN

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: 7.861143 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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

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Macromolecule #4: Nanobody 35

MacromoleculeName: Nanobody 35 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 17.057271 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKYLLPTAAA GLLLLAAQPA MAMQVQLQES GGGLVQPGGS LRLSCAASGF TFSNYKMNWV RQAPGKGLEW VSDISQSGAS ISYTGSVKG RFTISRDNAK NTLYLQMNSL KPEDTAVYYC ARCPAPFTRD CFDVTSTTYA YRGQGTQVTV SSHHHHHH

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Macromolecule #5: Psychosine receptor

MacromoleculeName: Psychosine receptor / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.697605 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNSTCIEEQH DLDHYLFPIV YIFVIIVSIP ANIGSLCVSF LQAKKESELG IYLFSLSLSD LLYALTLPLW IDYTWNKDNW TFSPALCKG SAFLMYMNFY SSTAFLTCIA VDRYLAVVYP LKFFFLRTRR FALMVSLSIW ILETIFNAVM LWEDETVVEY C DAEKSNFT ...String:
MNSTCIEEQH DLDHYLFPIV YIFVIIVSIP ANIGSLCVSF LQAKKESELG IYLFSLSLSD LLYALTLPLW IDYTWNKDNW TFSPALCKG SAFLMYMNFY SSTAFLTCIA VDRYLAVVYP LKFFFLRTRR FALMVSLSIW ILETIFNAVM LWEDETVVEY C DAEKSNFT LCYDKYPLEK WQINLNLFRT CTGYAIPLVT ILICNRKVYQ AVRHNKATEN KEKKRIIKLL VSITVIFVLC FT PFHVMLL IRCILEHAVN FEDHSNSGKR TYTMYRITVA LTSLNCVADP ILYCFVTETG RYDMWNILKF CTGRCNTSQR QRK RILSVS TKDTMELEVL EGRPLEVLFQ GPGSSGHHHH HH

UniProtKB: G-protein coupled receptor 65

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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: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Average electron dose: 60.0 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: 1.2 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 80542
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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