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- EMDB-44772: Cryo-EM structure of P2X3 receptor in complex with ATP:Mg2+ -

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Basic information

Entry
Database: EMDB / ID: EMD-44772
TitleCryo-EM structure of P2X3 receptor in complex with ATP:Mg2+
Map datastructure of P2X3 receptor in complex with ATP:Mg2+
Sample
  • Complex: Ternary complex of P2X3 receptor and ATP
    • Protein or peptide: P2X purinoceptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsP2X3 / camlipixant / cryo-EM / ion channel / receptor / MEMBRANE PROTEIN
Function / homology
Function and homology information


extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / response to ATP / postsynapse / ATP binding / plasma membrane
Similarity search - Function
P2X3 purinoceptor / : / ATP P2X receptors signature. / ATP P2X receptor / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
Biological speciesCanis lupus (gray wolf)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsThach T / Subramanian R
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biol Chem / Year: 2025
Title: Mechanistic insights into the selective targeting of P2X3 receptor by camlipixant antagonist.
Authors: Trung Thach / KanagaVijayan Dhanabalan / Prajwal Prabhakarrao Nandekar / Seth Stauffer / Iring Heisler / Sarah Alvarado / Jonathan Snyder / Ramaswamy Subramanian /
Abstract: ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts ...ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts to develop selective drugs targeting P2X3 have led to the development of camlipixant, a potent, selective P2X3 antagonist. However, the mechanisms of receptor desensitization, ion permeation, and structural basis of camlipixant binding to P2X3 remain unclear. Here, we report a cryo-EM structure of camlipixant-bound P2X3, revealing a previously undiscovered selective drug-binding site in the receptor. Our findings also demonstrate that conformational changes in the upper body domain, including the turret and camlipixant-binding pocket, play a critical role: turret opening facilitates P2X3 channel closure to a radius of 0.7 Å, hindering cation transfer, whereas turret closure leads to channel opening. Structural and functional studies combined with molecular dynamics simulations provide a comprehensive understanding of camlipixant's selective inhibition of P2X3, offering a foundation for future drug development targeting this receptor.
History
DepositionMay 7, 2024-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44772.png

Downloads & links

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Map

FileDownload / File: emd_44772.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of P2X3 receptor in complex with ATP:Mg2+
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 269.824 Å		1.05 Å/pix.
x 256 pix.
= 269.824 Å		1.05 Å/pix.
x 256 pix.
= 269.824 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.054 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.98526615 - 1.4350569
Average (Standard dev.)0.0024859933 (±0.028877297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 269.824 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_44772_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_44772_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of P2X3 receptor and ATP

EntireName: Ternary complex of P2X3 receptor and ATP
Components
  • Complex: Ternary complex of P2X3 receptor and ATP
    • Protein or peptide: P2X purinoceptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Ternary complex of P2X3 receptor and ATP

SupramoleculeName: Ternary complex of P2X3 receptor and ATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: P2X3 receptor and ATP
Source (natural)Organism: Canis lupus (gray wolf)
Molecular weightTheoretical: 126 KDa

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Macromolecule #1: P2X purinoceptor

MacromoleculeName: P2X purinoceptor / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Canis lupus (gray wolf)
Molecular weightTheoretical: 40.47752 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSDFFTYETT KSVVVKSWTI GVINRAVQLL IISYFVGWVF LHEKAYQVRD TAIESSVVTK VKGFGRYANR VMDVSDYVTP PQGTSVFVI ITKMIVTENQ MQGFCPESEE KYRCVSDSQC GPQRFPGGGI LTGRCVNYSS TLRTCEIQGW CPTEVDTVEM P VMMEAENF ...String:
GSDFFTYETT KSVVVKSWTI GVINRAVQLL IISYFVGWVF LHEKAYQVRD TAIESSVVTK VKGFGRYANR VMDVSDYVTP PQGTSVFVI ITKMIVTENQ MQGFCPESEE KYRCVSDSQC GPQRFPGGGI LTGRCVNYSS TLRTCEIQGW CPTEVDTVEM P VMMEAENF TIFIKNSIRF PLFNFEKGNL LPNLTAADMK TCRFHPDKAP FCPILRVGDV VKFAGQDFAK LARTGGVLGI KI GWVCDLD RAWDQCIPKY SFTRLDGVSE KSSVSPGYNF RFAKYYKMEN GSEYRTLLKA FGIRFDVLVY GNAGKFNIIP TII SSVAAF TSVGVGTVLC DIILLNFLKG ADQYKAKKFE EVDET

UniProtKB: P2X purinoceptor

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 3 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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

Concentration0.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMNaClSodium Chloride

Details: 20 mM HEPES, 150 mM NaCl, 0.08%DDM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 11
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: vitrification.
DetailsP2X3:ATP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Number grids imaged: 1 / Number real images: 60 / Average exposure time: 1.8 sec. / Average electron dose: 56.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.1 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 87363
Startup modelType of model: INSILICO MODEL / In silico model: ab initio construction
Final reconstructionNumber classes used: 50 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 44237
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 50
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Chain ID: A / Chain - Residue range: 1-361 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: The initial model consisted of monomer
Detailsreal refinement was done using Phenix
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9bpd:
Cryo-EM structure of P2X3 receptor in complex with ATP:Mg2+

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