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- PDB-8tra: Cryo-EM structure of the rat P2X7 receptor in complex with the al... -

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Basic information

Entry
Database: PDB / ID: 8tra
TitleCryo-EM structure of the rat P2X7 receptor in complex with the allosteric antagonist GSK1482160
ComponentsP2X purinoceptor 7
KeywordsMEMBRANE PROTEIN / Ion Channel / Ligand-gate Ion Channel / P2X Receptor / Allosteric Antagonist / High-Affinity Agonist
Function / homology
Function and homology information


The NLRP3 inflammasome / regulation of presynaptic dense core granule exocytosis / Platelet homeostasis / positive regulation of lymphocyte apoptotic process / positive regulation of bleb assembly / NAD transport / phagolysosome assembly / Elevation of cytosolic Ca2+ levels / phospholipid transfer to membrane / positive regulation of cytoskeleton organization ...The NLRP3 inflammasome / regulation of presynaptic dense core granule exocytosis / Platelet homeostasis / positive regulation of lymphocyte apoptotic process / positive regulation of bleb assembly / NAD transport / phagolysosome assembly / Elevation of cytosolic Ca2+ levels / phospholipid transfer to membrane / positive regulation of cytoskeleton organization / lymphocyte apoptotic process / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / gamma-aminobutyric acid secretion / pore complex assembly / positive regulation of interleukin-1 alpha production / plasma membrane organization / positive regulation of gamma-aminobutyric acid secretion / bleb / collagen metabolic process / negative regulation of cell volume / plasma membrane phospholipid scrambling / ATP export / T cell apoptotic process / response to fluid shear stress / positive regulation of prostaglandin secretion / bleb assembly / mitochondrial depolarization / vesicle budding from membrane / ceramide biosynthetic process / positive regulation of T cell apoptotic process / programmed cell death / prostaglandin secretion / positive regulation of ossification / cellular response to dsRNA / cell volume homeostasis / glutamate secretion / positive regulation of glutamate secretion / negative regulation of bone resorption / skeletal system morphogenesis / phospholipid translocation / positive regulation of macrophage cytokine production / response to ATP / response to zinc ion / positive regulation of mitochondrial depolarization / positive regulation of calcium ion transport into cytosol / T cell homeostasis / synaptic vesicle exocytosis / monoatomic cation transport / membrane protein ectodomain proteolysis / membrane depolarization / : / protein secretion / negative regulation of MAPK cascade / positive regulation of bone mineralization / neuronal action potential / response to electrical stimulus / response to mechanical stimulus / regulation of sodium ion transport / T cell proliferation / homeostasis of number of cells within a tissue / extrinsic apoptotic signaling pathway / release of sequestered calcium ion into cytosol / sensory perception of pain / reactive oxygen species metabolic process / protein serine/threonine kinase activator activity / positive regulation of glycolytic process / positive regulation of interleukin-1 beta production / positive regulation of protein secretion / establishment of localization in cell / mitochondrion organization / apoptotic signaling pathway / response to bacterium / lipopolysaccharide binding / protein catabolic process / neuromuscular junction / : / terminal bouton / T cell mediated cytotoxicity / cell morphogenesis / protein processing / calcium ion transmembrane transport / positive regulation of interleukin-6 production / positive regulation of T cell mediated cytotoxicity / response to calcium ion / calcium ion transport / MAPK cascade / cell-cell junction / nuclear envelope / channel activity / signaling receptor activity / scaffold protein binding / gene expression / response to lipopolysaccharide / positive regulation of MAPK cascade / cell surface receptor signaling pathway / postsynapse / defense response to Gram-positive bacterium
Similarity search - Function
P2X purinoreceptor 7, intracellular domain / P2X purinoreceptor 7 intracellular domain / P2X7 purinoceptor / : / ATP P2X receptors signature. / ATP P2X receptor / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / : / PALMITIC ACID / P2X purinoceptor 7
Similarity search - Component
Biological speciesRattus (rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.41 Å
AuthorsOken, A.C. / Ditter, I.A. / Lisi, N.E. / Krishnamurthy, I. / McCarthy, A.E. / Godsey, M.H. / Mansoor, S.E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R00HL138129 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM149551 United States
CitationJournal: Sci Adv / Year: 2024
Title: P2X receptors exhibit at least three modes of allosteric antagonism.
Authors: Adam C Oken / Ismayn A Ditter / Nicolas E Lisi / Ipsita Krishnamurthy / Michael H Godsey / Steven E Mansoor /
Abstract: P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A ...P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A number of small-molecule antagonists have been identified for these important pharmaceutical targets. However, the molecular pharmacology of P2X receptors is poorly understood because of the chemically disparate nature of antagonists and their differential actions on the seven constituent subtypes. Here, we report high-resolution cryo-electron microscopy structures of the homomeric rat P2X receptor bound to five previously known small-molecule allosteric antagonists and a sixth antagonist that we identify. Our structural, biophysical, and electrophysiological data define the molecular determinants of allosteric antagonism in this pharmacologically relevant receptor, revealing three distinct classes of antagonists that we call shallow, deep, and starfish. Starfish binders, exemplified by the previously unidentified antagonist methyl blue, represent a unique class of inhibitors with distinct functional properties that could be exploited to develop potent P2X ligands with substantial clinical impact.
History
DepositionAug 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: P2X purinoceptor 7
B: P2X purinoceptor 7
C: P2X purinoceptor 7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,34037
Polymers205,4173
Non-polymers7,92334
Water3,531196
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein / Sugars , 2 types, 9 molecules ABC

#1: Protein P2X purinoceptor 7


Mass: 68472.461 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus (rat) / Gene: P2rx7 / Cell line (production host): HEK293 GNTI- / Production host: Rattus (rat) / References: UniProt: Q64663
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 6 types, 224 molecules

#2: Chemical ChemComp-KF0 / N-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-L-prolinamide


Mass: 334.721 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C14H14ClF3N2O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 15
Source method: isolated from a genetically manipulated source
Formula: C16H32O2 / Source: (gene. exp.) Rattus (rat) / Cell line (production host): HEK293 GNTI- / Production host: Rattus (rat) / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 196 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Membrane protein / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Rattus (rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 GNTI-
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8208
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 394113 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214094
ELECTRON MICROSCOPYf_angle_d0.50719080
ELECTRON MICROSCOPYf_dihedral_angle_d6.6142037
ELECTRON MICROSCOPYf_chiral_restr0.042031
ELECTRON MICROSCOPYf_plane_restr0.0032385

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