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- PDB-9c48: Cryo-EM structure of the full-length human P2X4 receptor in the A... -

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Basic information

Entry
Database: PDB / ID: 9c48
TitleCryo-EM structure of the full-length human P2X4 receptor in the ATP-bound desensitized state
ComponentsP2X purinoceptor 4
KeywordsMEMBRANE PROTEIN / Ion Channel / Ligand-gated Ion Channel / P2X Receptor / Allosteric Antagonist
Function / homology
Function and homology information


Platelet homeostasis / sensory perception of touch / positive regulation of microglial cell migration / purinergic nucleotide receptor signaling pathway / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / ligand-gated calcium channel activity / Elevation of cytosolic Ca2+ levels / negative regulation of cardiac muscle hypertrophy / positive regulation of prostaglandin secretion ...Platelet homeostasis / sensory perception of touch / positive regulation of microglial cell migration / purinergic nucleotide receptor signaling pathway / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / ligand-gated calcium channel activity / Elevation of cytosolic Ca2+ levels / negative regulation of cardiac muscle hypertrophy / positive regulation of prostaglandin secretion / regulation of chemotaxis / response to fluid shear stress / tissue homeostasis / positive regulation of calcium ion transport / endothelial cell activation / positive regulation of endothelial cell chemotaxis / cellular response to zinc ion / relaxation of cardiac muscle / cellular response to ATP / response to ATP / parallel fiber to Purkinje cell synapse / positive regulation of calcium ion transport into cytosol / behavioral response to pain / membrane depolarization / positive regulation of blood vessel endothelial cell migration / response to axon injury / regulation of cardiac muscle contraction / neuronal action potential / Purinergic signaling in leishmaniasis infection / regulation of sodium ion transport / sensory perception of pain / positive regulation of calcium-mediated signaling / response to ischemia / apoptotic signaling pathway / calcium-mediated signaling / postsynaptic density membrane / terminal bouton / calcium ion transmembrane transport / regulation of blood pressure / positive regulation of nitric oxide biosynthetic process / cell junction / cell body / monoatomic ion transmembrane transport / dendritic spine / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cadherin binding / copper ion binding / signaling receptor binding / lysosomal membrane / neuronal cell body / perinuclear region of cytoplasm / glutamatergic synapse / signal transduction / extracellular exosome / zinc ion binding / ATP binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
P2X4 purinoceptor / : / ATP P2X receptors signature. / ATP P2X receptor / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / P2X purinoceptor 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsShi, H. / Ditter, I.A. / Oken, A.C. / 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: 2025
Title: Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket.
Authors: Haoyuan Shi / Ismayn A Ditter / Adam C Oken / Steven E Mansoor /
Abstract: P2X receptors (P2XRs) are adenosine 5'-triphosphate (ATP)-gated ion channels comprising homomeric and heteromeric trimers of seven subtypes (P2X1-P2X7) that confer different rates of desensitization. ...P2X receptors (P2XRs) are adenosine 5'-triphosphate (ATP)-gated ion channels comprising homomeric and heteromeric trimers of seven subtypes (P2X1-P2X7) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-electron microscopy structures of full-length wild-type human P2X4 receptor in apo closed, antagonist-bound inhibited, and ATP-bound desensitized states. Because the apo closed and antagonist-bound inhibited state structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the ATP-bound desensitized state structure does not, the cytoplasmic cap is formed before agonist binding. Furthermore, structural and functional data suggest the cytoplasmic cap is stabilized by lipids to modulate desensitization, and P2X4 is modified by glycosylation and palmitoylation. Last, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that facilitates development of small-molecule modulators.
History
DepositionJun 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release
Revision 1.0Jan 29, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1May 14, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: P2X purinoceptor 4
B: P2X purinoceptor 4
C: P2X purinoceptor 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,13627
Polymers130,2453
Non-polymers7,89124
Water4,486249
1


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

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein P2X purinoceptor 4 / P2X4 / ATP receptor / Purinergic receptor


Mass: 43415.000 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: P2RX4 / Cell (production host): HEK293 GNTI- / Production host: Homo sapiens (human) / References: UniProt: Q99571

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Sugars , 3 types, 18 molecules

#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#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.: 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 , 3 types, 255 molecules

#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 249 / 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: Homo sapiens (human)
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: TFS 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: 1500 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 15582
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122405 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058409
ELECTRON MICROSCOPYf_angle_d0.71311466
ELECTRON MICROSCOPYf_dihedral_angle_d15.3813006
ELECTRON MICROSCOPYf_chiral_restr0.0541395
ELECTRON MICROSCOPYf_plane_restr0.0041404

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