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- EMDB-44370: Cryo-EM structure of the closed NF449-bound human P2X1 receptor -

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Basic information

Entry
Database: EMDB / ID: EMD-44370
TitleCryo-EM structure of the closed NF449-bound human P2X1 receptor
Map dataMap of the P2X1 receptor generated in Cryosparc. (J549 non-uniform refinement job)
Sample
  • Complex: P2X1 receptor trimer
    • Protein or peptide: P2X purinoceptor 1
  • Ligand: 4,4',4'',4'''-{carbonylbis[azanediylbenzene-5,1,3-triylbis(carbonylazanediyl)]}tetra(benzene-1,3-disulfonic acid)
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
KeywordsIon channel / trimer / NF449-bound / closed. / MEMBRANE PROTEIN
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
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsFelix MB / Alisa G / Hariprasad V / Jesse IM / David MT
Funding support Australia, 1 items
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, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44370.png

Downloads & links

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Map

FileDownload / File: emd_44370.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the P2X1 receptor generated in Cryosparc. (J549 non-uniform refinement job)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 240 pix.
= 196.8 Å		0.82 Å/pix.
x 240 pix.
= 196.8 Å		0.82 Å/pix.
x 240 pix.
= 196.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.2586453 - 0.57092595
Average (Standard dev.)0.000029940276 (±0.019361824)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 196.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44370_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the P2X1 receptor generated in...

Fileemd_44370_half_map_1.map
AnnotationHalf map of the P2X1 receptor generated in Cryosparc. (J549 non-uniform refinement job)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the P2X1 receptor generated in...

Fileemd_44370_half_map_2.map
AnnotationHalf map of the P2X1 receptor generated in Cryosparc. (J549 non-uniform refinement job)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : P2X1 receptor trimer

EntireName: P2X1 receptor trimer
Components
  • Complex: P2X1 receptor trimer
    • Protein or peptide: P2X purinoceptor 1
  • Ligand: 4,4',4'',4'''-{carbonylbis[azanediylbenzene-5,1,3-triylbis(carbonylazanediyl)]}tetra(benzene-1,3-disulfonic acid)
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: P2X1 receptor trimer

SupramoleculeName: P2X1 receptor trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 134.94 KDa

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Macromolecule #1: P2X purinoceptor 1

MacromoleculeName: P2X purinoceptor 1 / type: protein_or_peptide / ID: 1
Details: Partial sections of the N and C-termini remain unmodelled due to insufficient cryo-EM density.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.038957 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MARRFQEELA AFLFEYDTPR MVLVRNKKVG VIFRLIQLVV LVYVIGWVFL YEKGYQTSSG LISSVSVKLK GLAVTQLPGL GPQVWDVAD YVFPAQGDNS FVVMTNFIVT PKQTQGYCAE HPEGGICKED SGCTPGKAKR KAQGIRTGKC VAFNDTVKTC E IFGWCPVE ...String:
MARRFQEELA AFLFEYDTPR MVLVRNKKVG VIFRLIQLVV LVYVIGWVFL YEKGYQTSSG LISSVSVKLK GLAVTQLPGL GPQVWDVAD YVFPAQGDNS FVVMTNFIVT PKQTQGYCAE HPEGGICKED SGCTPGKAKR KAQGIRTGKC VAFNDTVKTC E IFGWCPVE VDDDIPRPAL LREAENFTLF IKNSISFPRF KVNRRNLVEE VNAAHMKTCL FHKTLHPLCP VFQLGYVVQE SG QNFSTLA EKGGVVGITI DWHCDLDWHV RHCRPIYEFH GLYEEKNLSP GFNFRFARHF VENGTNYRHL FKVFGIRFDI LVD GKAGKF DIIPTMTTIG SGIGIFGVAT VLCDLLLLHI LPKRHYYKQK KFKYAEDMGP GAAERDLAAT SSTLGLQENM RTS

UniProtKB: P2X purinoceptor 1

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Macromolecule #2: 4,4',4'',4'''-{carbonylbis[azanediylbenzene-5,1,3-triylbis(carbon...

MacromoleculeName: 4,4',4'',4'''-{carbonylbis[azanediylbenzene-5,1,3-triylbis(carbonylazanediyl)]}tetra(benzene-1,3-disulfonic acid)
type: ligand / ID: 2 / Number of copies: 3 / Formula: A1ALI
Molecular weightTheoretical: 1.329236 KDa

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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: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 18 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration14.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
100.0 mMNaClSodium Chloride
0.01 %C47H88O22Lauryl Maltose Neopentyl Glycol
0.0006 %C31H50O4Cholesteryl hemisuccinate
0.1 mMC41H24N6Na8O29S8NF449
0.3 mMC13H17F13NO4PFluorinated Fos-Choline-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.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: OTHER / Details: Model used: Glow Discharge Peelco Easyglow
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
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

MicroscopeTFS KRIOS
DetailsInitial grid screening was conducted using a 200kV TFS Artica cryo-electron microscope prior to imaging on a Titan Krios cryo-electron microscope.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5837 / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3837432
Startup modelType of model: NONE
Details: The initial particle set was employed to create an ab initio model, which was then utilised for subsequent 3D refinement tasks.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1.4) / Software - details: Non-uniform refinement job / Number images used: 41932
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.4) / Software - details: Ab initio job
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.4) / Software - details: Heterogeneous refinement job
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 1.4) / Software - details: Heterogeneous refinement job
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1575


Chain - Residue range: 1-399 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: Full human P2X1 receptor trimer
DetailsInitial fitting was performed in Chimerax and then refinements were performed in Coot and Phenix.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 72.9
Output model

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

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