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- EMDB-44299: Cryo-EM structure of the desensitised ATP-bound human P2X1 receptor -

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Basic information

Entry
Database: EMDB / ID: EMD-44299
TitleCryo-EM structure of the desensitised ATP-bound human P2X1 receptor
Map dataMap of the P2X1 receptor generated in Cryosparc. (J609 homogenous refinement job)
Sample
  • Complex: P2X1 receptor trimer
    • Protein or peptide: P2X purinoceptor 1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsIon channel / trimer / ATP-bound / desensitised / 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 / Elevation of cytosolic Ca2+ levels / regulation of presynaptic cytosolic calcium ion concentration ...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 / Elevation of cytosolic Ca2+ levels / regulation of presynaptic cytosolic calcium ion concentration / 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: 1.96 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: Bennetts FM / Venugopal H / Glukhova A / Mobbs JI / Ventura S / Thal DM
History
DepositionMar 27, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44299.png

Downloads & links

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Map

FileDownload / File: emd_44299.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. (J609 homogenous 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: 0.27
Minimum - Maximum-0.6462056 - 2.4074166
Average (Standard dev.)-0.0007459086 (±0.063122116)
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_44299_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharp map of the P2X1 receptor generated in...

Fileemd_44299_additional_1.map
AnnotationSharp map of the P2X1 receptor generated in Phenix. (Generated from original cryo-EM 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_44299_half_map_1.map
AnnotationHalf map of the P2X1 receptor generated in Cryosparc. (J609 homogenous 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_44299_half_map_2.map
AnnotationHalf map of the P2X1 receptor generated in Cryosparc. (J609 homogenous 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: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • 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 / 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: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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: 9 / 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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 66 / 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

Concentration19 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
100.0 mMNaClSodium Chloride
0.01 %C47H88O22Lauryl Maltose Neopentyl Glycol
0.0006 %C31H50O4Cholesteryl hemisuccinate
1.0 mMC10H16N5O13P3ATP
1.0 mMMG2+Magnesium ion
0.4 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 1 mM ATP and 1 mM MgCl2 overnight before vitrification. Additionally, 0.4 mM or 1.2 mM of ...Details: The buffer consists of 50 mM Tris (pH 8), 100 mM NaCl, 0.01% LMNG, and 0.0006% CHS supplemented with 1 mM ATP and 1 mM MgCl2 overnight before vitrification. Additionally, 0.4 mM or 1.2 mM of 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: 2 / Number real images: 10756 / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Details: 4578 images were collected on the grid containing P2X1 receptor supplemented with a higher concentration of fluor-FC8 (1.2 mM), while 6178 images were collected on the grid with a lower ...Details: 4578 images were collected on the grid containing P2X1 receptor supplemented with a higher concentration of fluor-FC8 (1.2 mM), while 6178 images were collected on the grid with a lower concentration of fluor-FC8 (0.4 mM)
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: 5312810
Details: Initially, 2,565,511 particles were picked for the higher concentration fluor-FC8 P2X1 receptor sample, and 2,747,299 particles were picked for the lower concentration sample.
Startup modelType of model: NONE
Details: Initial particle set was used to generate an ab initio model which was subsequently used for 3D refinement jobs
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1.3) / Software - details: Homogenous refinement job / Number images used: 481309
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.3) / Software - details: Ab initio job
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.3) / Software - details: Heterogeneous refinement job
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 1.3) / 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: 60.7
Output model

PDB-9b73:
Cryo-EM structure of the desensitised ATP-bound human P2X1 receptor

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