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- EMDB-44548: Human proton sensing receptor GPR65 in complex with miniGs - focu... -

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Basic information

Entry
Database: EMDB / ID: EMD-44548
TitleHuman proton sensing receptor GPR65 in complex with miniGs - focused map of 7TM
Map data
Sample
  • Complex: GPR65 receptor in complex with miniGs
    • Complex: GPR65 receptor
    • Complex: miniGs (alpha and beta subunits)
    • Complex: Nanobody 35
KeywordsReceptor / Proton-sensor / G protein / SIGNALING PROTEIN
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsHoward MK / Hoppe N / Huang XP / Macdonald CB / Mehrotra E / Rockefeller Grimes P / Zahm AM / Trinidad DD / English J / Coyote-Maestas W / Manglik A
Funding support United States, 1 items
OrganizationGrant numberCountry
Chan Zuckerberg Initiative United States
CitationJournal: Cell / Year: 2025
Title: Molecular basis of proton sensing by G protein-coupled receptors.
Authors: Matthew K Howard / Nicholas Hoppe / Xi-Ping Huang / Darko Mitrovic / Christian B Billesbølle / Christian B Macdonald / Eshan Mehrotra / Patrick Rockefeller Grimes / Donovan D Trinidad / ...Authors: Matthew K Howard / Nicholas Hoppe / Xi-Ping Huang / Darko Mitrovic / Christian B Billesbølle / Christian B Macdonald / Eshan Mehrotra / Patrick Rockefeller Grimes / Donovan D Trinidad / Lucie Delemotte / Justin G English / Willow Coyote-Maestas / Aashish Manglik /
Abstract: Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. ...Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating ∼9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.
History
DepositionApr 21, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44548.png

Downloads & links

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Map

FileDownload / File: emd_44548.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 300 pix.
= 250.5 Å		0.84 Å/pix.
x 300 pix.
= 250.5 Å		0.84 Å/pix.
x 300 pix.
= 250.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum-15.436617 - 28.217102000000001
Average (Standard dev.)-0.024676943 (±0.3986081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 250.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_44548_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44548_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44548_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GPR65 receptor in complex with miniGs

EntireName: GPR65 receptor in complex with miniGs
Components
  • Complex: GPR65 receptor in complex with miniGs
    • Complex: GPR65 receptor
    • Complex: miniGs (alpha and beta subunits)
    • Complex: Nanobody 35

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Supramolecule #1: GPR65 receptor in complex with miniGs

SupramoleculeName: GPR65 receptor in complex with miniGs / type: complex / ID: 1 / Parent: 0

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Supramolecule #2: GPR65 receptor

SupramoleculeName: GPR65 receptor / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: miniGs (alpha and beta subunits)

SupramoleculeName: miniGs (alpha and beta subunits) / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Nanobody 35

SupramoleculeName: Nanobody 35 / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Lama glama (llama)

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 411592
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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