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- EMDB-63219: Cryo-EM structure of human apo inactive GPR4 -

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Basic information

Entry
Database: EMDB / ID: EMD-63219
TitleCryo-EM structure of human apo inactive GPR4
Map data
Sample
  • Organelle or cellular component: inactive GPR4
    • Protein or peptide: Heavy chain of anti-Bril Fab
    • Protein or peptide: anti-Fab Nanobody
    • Protein or peptide: Light chain of anti-Bril Fab
    • Protein or peptide: G-protein coupled receptor 4,Soluble cytochrome b562
KeywordsGPCR / class A / GPR4 / cryo-EM / protein sensing / inactive state / MEMBRANE PROTEIN/IMMUNE SYSTEM / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


glomerular mesangial cell development / regulation of vascular permeability / response to acidic pH / Class A/1 (Rhodopsin-like receptors) / angiogenesis involved in wound healing / positive regulation of Rho protein signal transduction / regulation of cell adhesion / negative regulation of angiogenesis / electron transport chain / G protein-coupled receptor activity ...glomerular mesangial cell development / regulation of vascular permeability / response to acidic pH / Class A/1 (Rhodopsin-like receptors) / angiogenesis involved in wound healing / positive regulation of Rho protein signal transduction / regulation of cell adhesion / negative regulation of angiogenesis / electron transport chain / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of inflammatory response / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / electron transfer activity / periplasmic space / iron ion binding / G protein-coupled receptor signaling pathway / heme binding / plasma membrane
Similarity search - Function
G protein-coupled receptor 4 orphan / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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)
Similarity search - Domain/homology
Soluble cytochrome b562 / G-prodeshotein coupled receptor 4
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChen LN / Zhou H / Xi K / Cheng SZ / Liu YF / Fu YF / Ma XY / Xu P / Ji SY / Wang WW ...Chen LN / Zhou H / Xi K / Cheng SZ / Liu YF / Fu YF / Ma XY / Xu P / Ji SY / Wang WW / Shen DD / Zhang HB / Shen QY / Chai R / Zhang M / Yang L / Han F / Mao CY / Cai XJ / Zhang Y
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2025
Title: Proton perception and activation of a proton-sensing GPCR.
Authors: Li-Nan Chen / Hui Zhou / Kun Xi / Shizhuo Cheng / Yongfeng Liu / Yifan Fu / Xiangyu Ma / Ping Xu / Su-Yu Ji / Wei-Wei Wang / Dan-Dan Shen / Huibing Zhang / Qingya Shen / Renjie Chai / Min ...Authors: Li-Nan Chen / Hui Zhou / Kun Xi / Shizhuo Cheng / Yongfeng Liu / Yifan Fu / Xiangyu Ma / Ping Xu / Su-Yu Ji / Wei-Wei Wang / Dan-Dan Shen / Huibing Zhang / Qingya Shen / Renjie Chai / Min Zhang / Lin Yang / Feng Han / Chunyou Mao / Xiujun Cai / Yan Zhang /
Abstract: Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. ...Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. However, the molecular mechanism of proton sensing and activation of these receptors remains elusive. Here, we present cryoelectron microscopy (cryo-EM) structures of human GPR4, a prototypical proton-sensing GPCR, in its inactive and active states. Our studies reveal that three extracellular histidine residues are crucial for proton sensing of human GPR4. The binding of protons induces substantial conformational changes in GPR4's ECLs, particularly in ECL2, which transforms from a helix-loop to a β-turn-β configuration. This transformation leads to the rearrangements of H-bond network and hydrophobic packing, relayed by non-canonical motifs to accommodate G proteins. Furthermore, the antagonist NE52-QQ57 hinders human GPR4 activation by preventing hydrophobic stacking rearrangement. Our findings provide a molecular framework for understanding the activation mechanism of a human proton-sensing GPCR, aiding future drug discovery.
History
DepositionJan 19, 2025-
Header (metadata) releaseJul 9, 2025-
Map releaseJul 9, 2025-
UpdateJan 14, 2026-
Current statusJan 14, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63219.png

Downloads & links

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Map

FileDownload / File: emd_63219.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)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.24
Minimum - Maximum-0.60029393 - 1.4150951
Average (Standard dev.)-0.0025912148 (±0.03761162)
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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Half map: #2

Fileemd_63219_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63219_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : inactive GPR4

EntireName: inactive GPR4
Components
  • Organelle or cellular component: inactive GPR4
    • Protein or peptide: Heavy chain of anti-Bril Fab
    • Protein or peptide: anti-Fab Nanobody
    • Protein or peptide: Light chain of anti-Bril Fab
    • Protein or peptide: G-protein coupled receptor 4,Soluble cytochrome b562

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Supramolecule #1: inactive GPR4

SupramoleculeName: inactive GPR4 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Heavy chain of anti-Bril Fab

MacromoleculeName: Heavy chain of anti-Bril Fab / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.00885 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHT

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Macromolecule #2: anti-Fab Nanobody

MacromoleculeName: anti-Fab Nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.159438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGRTIS RYAMSWFRQA PGKEREFVAV ARRSGDGAFY ADSVQGRFTV SRDDAKNTVY LQMNSLKPE DTAVYYCAID SDTFYSGSYD YWGQGTQVTV S

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Macromolecule #3: Light chain of anti-Bril Fab

MacromoleculeName: Light chain of anti-Bril Fab / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.586205 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

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

MacromoleculeName: G-protein coupled receptor 4,Soluble cytochrome b562 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 53.012133 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGNHTWEGCH VDSRVDHLFP PSLYIFVIGV GLPTNCLALW AAYRQVQQRN ELGVYLMNLS IADLLYICTL PLWVDYFLHH DNWIHGPGS CKLFGFIFYT NIYISIAFLC CISVDRYLAV AHPLRFARLR RVKTAVAVSS VVWATELGAN SAPLFHDELF R DRYNHTFC ...String:
MGNHTWEGCH VDSRVDHLFP PSLYIFVIGV GLPTNCLALW AAYRQVQQRN ELGVYLMNLS IADLLYICTL PLWVDYFLHH DNWIHGPGS CKLFGFIFYT NIYISIAFLC CISVDRYLAV AHPLRFARLR RVKTAVAVSS VVWATELGAN SAPLFHDELF R DRYNHTFC FEKFPMEGWV AWMNLYRVFV GFLFPWALML LSYRGILRAV EKRRADLEDN WETLNDNLKV IEKADNAAQV KD ALTKMRA AALDAQKATD KSPDSPEMKD FRHGFDILVG QIDDALKLAN EGKVKEAQAA AEQLKTTRNA YIQKYLDWER ERL KRLERI KRLALSLIAI VLVCFAPYHV LLLSRSAIYL GRPWDCGFEE RVFSAYHSSL AFTSLNCVAD PILYCLVNEG ARSD VAKAL HNLLRFLASD KPQEMANASL TLETPLTSKR NSTAKAMTGS WAATPPSQGD QVQLKMLPPA Q

UniProtKB: G-prodeshotein coupled receptor 4, Soluble cytochrome b562, G-prodeshotein 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS 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 / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7000000000000001 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 261077
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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