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- PDB-8v4s: Cryo-EM structure of the rat P2X7 receptor in the apo closed stat... -

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Basic information

Entry
Database: PDB / ID: 8v4s
TitleCryo-EM structure of the rat P2X7 receptor in the apo closed state purified in the absence of sodium
ComponentsP2X purinoceptor 7
KeywordsMEMBRANE PROTEIN / Ion Channel / Ligand-gate Ion Channel / P2X Receptor / Allosteric Antagonist / High-Affinity Agonist
Function / homology
Function and homology information


Platelet homeostasis / The NLRP3 inflammasome / regulation of presynaptic dense core granule exocytosis / 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 ...Platelet homeostasis / The NLRP3 inflammasome / regulation of presynaptic dense core granule exocytosis / 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 / purinergic nucleotide receptor signaling pathway / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / lymphocyte apoptotic process / positive regulation of monoatomic ion transmembrane transport / gamma-aminobutyric acid secretion / pore complex assembly / positive regulation of interleukin-1 alpha production / plasma membrane organization / negative regulation of cell volume / positive regulation of gamma-aminobutyric acid secretion / ATP export / bleb / collagen metabolic process / plasma membrane phospholipid scrambling / response to fluid shear stress / T cell apoptotic process / positive regulation of prostaglandin secretion / bleb assembly / positive regulation of T cell apoptotic process / mitochondrial depolarization / vesicle budding from membrane / prostaglandin secretion / ceramide biosynthetic process / cellular response to dsRNA / programmed cell death / positive regulation of glutamate secretion / positive regulation of ossification / cell volume homeostasis / glutamate secretion / skeletal system morphogenesis / negative regulation of bone resorption / phospholipid translocation / positive regulation of macrophage cytokine production / positive regulation of calcium ion transport into cytosol / response to ATP / positive regulation of mitochondrial depolarization / response to zinc ion / T cell homeostasis / cellular response to organic cyclic compound / monoatomic cation transport / synaptic vesicle exocytosis / membrane depolarization / membrane protein ectodomain proteolysis / neuronal action potential / protein secretion / negative regulation of MAPK cascade / response to electrical stimulus / positive regulation of bone mineralization / response to mechanical stimulus / T cell proliferation / regulation of sodium ion transport / extrinsic apoptotic signaling pathway / release of sequestered calcium ion into cytosol / homeostasis of number of cells within a tissue / sensory perception of pain / positive regulation of glycolytic process / reactive oxygen species metabolic process / protein serine/threonine kinase activator activity / positive regulation of interleukin-1 beta production / mitochondrion organization / establishment of localization in cell / positive regulation of protein secretion / apoptotic signaling pathway / calcium ion transmembrane transport / lipopolysaccharide binding / response to bacterium / neuromuscular junction / cell morphogenesis / protein catabolic process / response to organic cyclic compound / terminal bouton / T cell mediated cytotoxicity / protein processing / positive regulation of interleukin-6 production / response to calcium ion / channel activity / positive regulation of T cell mediated cytotoxicity / calcium ion transport / MAPK cascade / cell-cell junction / nuclear envelope / signaling receptor activity / gene expression / scaffold protein binding / postsynapse / response to lipopolysaccharide / positive regulation of MAPK cascade / cell surface receptor signaling pathway / 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: 2.49 Å
AuthorsOken, A.C. / Lisi, N.E. / Krishnamurthy, I. / McCarthy, A.E. / Godsey, M.H. / Glasfeld, A. / 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: Nat Commun / Year: 2024
Title: High-affinity agonism at the P2X receptor is mediated by three residues outside the orthosteric pocket.
Authors: Adam C Oken / Nicolas E Lisi / Ipsita Krishnamurthy / Alanna E McCarthy / Michael H Godsey / Arthur Glasfeld / Steven E Mansoor /
Abstract: P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X is a potential experimental tool and ...P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.
History
DepositionNov 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _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)212,97736
Polymers205,4173
Non-polymers7,55933
Water4,161231
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, 12 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: Homo sapiens (human) / References: UniProt: Q64663
#4: 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.: 9 / 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 , 4 types, 255 molecules

#2: 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
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: 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) / Gene: P2rx7 / Cell line (production host): HEK293 GNTI- / Production host: Homo sapiens (human) / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 231 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 1400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 11353
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: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 273076 / Symmetry type: POINT

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