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- PDB-9b95: Cryo-EM structure of the closed NF449-bound human P2X1 receptor -

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Basic information

Entry
Database: PDB / ID: 9b95
TitleCryo-EM structure of the closed NF449-bound human P2X1 receptor
ComponentsP2X purinoceptor 1
KeywordsMEMBRANE PROTEIN / Ion channel / trimer / NF449-bound / closed.
Function / homology
Function and homology information


regulation of vascular associated smooth muscle contraction / Platelet homeostasis / insemination / positive regulation of calcium ion import across plasma membrane / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / suramin binding / serotonin secretion by platelet / regulation of presynaptic cytosolic calcium ion concentration / Elevation of cytosolic Ca2+ levels ...regulation of vascular associated smooth muscle contraction / Platelet homeostasis / insemination / positive regulation of calcium ion import across plasma membrane / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / suramin binding / serotonin secretion by platelet / regulation of presynaptic cytosolic calcium ion concentration / Elevation of cytosolic Ca2+ levels / ligand-gated calcium channel activity / ceramide biosynthetic process / regulation of synaptic vesicle exocytosis / response to ATP / neuronal action potential / specific granule membrane / monoatomic cation channel activity / monoatomic ion transport / presynaptic active zone membrane / secretory granule membrane / synaptic transmission, glutamatergic / calcium ion transmembrane transport / platelet activation / regulation of blood pressure / postsynaptic membrane / membrane raft / external side of plasma membrane / glutamatergic synapse / Neutrophil degranulation / protein-containing complex binding / apoptotic process / signal transduction / protein-containing complex / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
P2X1 purinoceptor / : / ATP P2X receptors signature. / ATP P2X receptor / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
: / P2X purinoceptor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsFelix, M.B. / Alisa, G. / Hariprasad, V. / Jesse, I.M. / David, M.T.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1196951 Australia
CitationJournal: Nat Commun / Year: 2024
Title: Structural insights into the human P2X1 receptor and ligand interactions.
Authors: Felix M Bennetts / Hariprasad Venugopal / Alisa Glukhova / Jesse I Mobbs / Sabatino Ventura / David M Thal /
Abstract: The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this ...The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this area has been hindered by limited structural information and a lack of well-characterised tool compounds. In this study, we employ cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [H]-α,β-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validate key ligand-receptor interactions and identify conserved and non-conserved residues critical for ligand binding or receptor modulation. This research expands our understanding of the P2X1 receptor structure at a molecular level and opens new avenues for in silico drug design targeting the P2X1 receptor.
History
DepositionApr 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Oct 30, 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 1
B: P2X purinoceptor 1
C: P2X purinoceptor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,7689
Polymers135,1173
Non-polymers4,6516
Water32418
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein P2X purinoceptor 1 / P2X1 / ATP receptor / Purinergic receptor


Mass: 45038.957 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Partial sections of the N and C-termini remain unmodelled due to insufficient cryo-EM density.
Source: (gene. exp.) Homo sapiens (human) / Gene: P2RX1, P2X1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P51575
#2: Chemical ChemComp-A1ALI / 4,4',4'',4'''-{carbonylbis[azanediylbenzene-5,1,3-triylbis(carbonylazanediyl)]}tetra(benzene-1,3-disulfonic acid)


Mass: 1329.236 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C41H32N6O29S8 / Feature type: SUBJECT OF INVESTIGATION
#3: 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.: 3 / 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
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Formula: H2O
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: P2X1 receptor trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.134940 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Details: The buffer consists of 50 mM Tris (pH 8), 100 mM NaCl, 0.01% LMNG, and 0.0006% CHS supplemented with 0.1 mM NF449. Additionally, 0.3 mM fluorinated Fos-Choline-8 (fluor-FC8) was added just ...Details: The buffer consists of 50 mM Tris (pH 8), 100 mM NaCl, 0.01% LMNG, and 0.0006% CHS supplemented with 0.1 mM NF449. Additionally, 0.3 mM fluorinated Fos-Choline-8 (fluor-FC8) was added just one minute prior to vitrification.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2100 mMSodium ChlorideNaCl1
30.01 %Lauryl Maltose Neopentyl GlycolC47H88O221
40.0006 %Cholesteryl hemisuccinateC31H50O41
50.1 mMNF449C41H24N6Na8O29S81
60.3 mMFluorinated Fos-Choline-8C13H17F13NO4P1
SpecimenConc.: 14.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Model used: Glow Discharge Peelco Easyglow / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Grids were frozen in liquid ethane using a Vitrobot Mark III operated at 4 degrees Celsius and 100% humidity with 12 blot force and 2 seconds blot time.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: Initial grid screening was conducted using a 200kV TFS Artica cryo-electron microscope prior to imaging on a Titan Krios cryo-electron microscope.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5837

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC1.4particle selectionTemplate picking job
4cryoSPARC1.4CTF correctionPatch CTF correction job
7Coot0.9.8.92model fitting
9PHENIX1.20.1model refinement
10cryoSPARC1.4initial Euler assignmentAb initio job
11cryoSPARC1.4final Euler assignmentHeterogeneous refinement job
12cryoSPARC1.4classificationHeterogeneous refinement job
13cryoSPARC1.43D reconstructionNon-uniform refinement job
CTF correctionDetails: CTF correction was initially conducted on the set of motion-corrected images, with subsequent local CTF corrections applied to the final subset of particles.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3837432
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41932 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 72.9 / Protocol: RIGID BODY FIT / Space: REAL
Details: Initial fitting was performed in Chimerax and then refinements were performed in Coot and Phenix.
Atomic model buildingAccession code: P51575 / Chain residue range: 1-399 / Details: Full human P2X1 receptor trimer / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 113.03 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00178064
ELECTRON MICROSCOPYf_angle_d0.678410998
ELECTRON MICROSCOPYf_chiral_restr0.04451173
ELECTRON MICROSCOPYf_plane_restr0.00311383
ELECTRON MICROSCOPYf_dihedral_angle_d9.88892826

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