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- EMDB-42976: Cryo-EM structure of the rat P2X7 receptor in the apo closed stat... -

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Basic information

Entry
Database: EMDB / ID: EMD-42976
TitleCryo-EM structure of the rat P2X7 receptor in the apo closed state purified in the absence of sodium
Map dataLocally sharpened map for APO rP2X7 purified in KCl
Sample
  • Complex: Membrane protein
    • Protein or peptide: P2X purinoceptor 7
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: PALMITIC ACID
  • Ligand: water
KeywordsMembrane Protein / Ion Channel / Ligand-gate Ion Channel / P2X Receptor / Allosteric Antagonist / High-Affinity Agonist
Function / homology
Function and homology information


The NLRP3 inflammasome / regulation of presynaptic dense core granule exocytosis / Platelet homeostasis / 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 ...The NLRP3 inflammasome / regulation of presynaptic dense core granule exocytosis / Platelet homeostasis / 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 activity / extracellularly ATP-gated monoatomic cation channel activity / lymphocyte apoptotic process / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / gamma-aminobutyric acid secretion / pore complex assembly / positive regulation of interleukin-1 alpha production / plasma membrane organization / positive regulation of gamma-aminobutyric acid secretion / negative regulation of cell volume / bleb / collagen metabolic process / plasma membrane phospholipid scrambling / ATP export / response to fluid shear stress / T cell apoptotic process / positive regulation of prostaglandin secretion / bleb assembly / mitochondrial depolarization / vesicle budding from membrane / ceramide biosynthetic process / positive regulation of T cell apoptotic process / programmed cell death / prostaglandin secretion / positive regulation of ossification / cellular response to dsRNA / cell volume homeostasis / positive regulation of glutamate secretion / glutamate secretion / negative regulation of bone resorption / skeletal system morphogenesis / phospholipid translocation / positive regulation of macrophage cytokine production / response to ATP / response to zinc ion / positive regulation of mitochondrial depolarization / positive regulation of calcium ion transport into cytosol / T cell homeostasis / synaptic vesicle exocytosis / monoatomic cation transport / membrane depolarization / membrane protein ectodomain proteolysis / : / protein secretion / negative regulation of MAPK cascade / neuronal action potential / positive regulation of bone mineralization / response to electrical stimulus / response to mechanical stimulus / regulation of sodium ion transport / T cell proliferation / homeostasis of number of cells within a tissue / extrinsic apoptotic signaling pathway / release of sequestered calcium ion into cytosol / sensory perception of pain / protein serine/threonine kinase activator activity / reactive oxygen species metabolic process / positive regulation of glycolytic process / positive regulation of interleukin-1 beta production / apoptotic signaling pathway / mitochondrion organization / positive regulation of protein secretion / establishment of localization in cell / response to bacterium / lipopolysaccharide binding / protein catabolic process / neuromuscular junction / terminal bouton / T cell mediated cytotoxicity / cell morphogenesis / : / protein processing / calcium ion transmembrane transport / response to calcium ion / positive regulation of interleukin-6 production / positive regulation of T cell mediated cytotoxicity / calcium ion transport / MAPK cascade / cell-cell junction / nuclear envelope / channel activity / signaling receptor activity / scaffold protein binding / gene expression / response to lipopolysaccharide / positive regulation of MAPK cascade / cell surface receptor signaling pathway / postsynapse / 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
Biological speciesRattus (rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsOken AC / Lisi NE / Krishnamurthy I / McCarthy AE / Godsey MH / Glasfeld A / Mansoor SE
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42976.png

Downloads & links

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Map

FileDownload / File: emd_42976.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally sharpened map for APO rP2X7 purified in KCl
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 600 pix.
= 388.8 Å		0.65 Å/pix.
x 600 pix.
= 388.8 Å		0.65 Å/pix.
x 600 pix.
= 388.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.648 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.0074512735 - 1.36883
Average (Standard dev.)0.00048488166 (±0.012710733)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 388.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42976_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map for APO rP2X7 purified in KCl

Fileemd_42976_additional_1.map
AnnotationSharpened map for APO rP2X7 purified in KCl
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map for APO rP2X7 purified in KCl

Fileemd_42976_additional_2.map
AnnotationUnsharpened map for APO rP2X7 purified in KCl
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A for APO rP2X7 purified in KCl

Fileemd_42976_half_map_1.map
AnnotationHalf map A for APO rP2X7 purified in KCl
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B for APO rP2X7 purified in KCl

Fileemd_42976_half_map_2.map
AnnotationHalf map B for APO rP2X7 purified in KCl
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Membrane protein

EntireName: Membrane protein
Components
  • Complex: Membrane protein
    • Protein or peptide: P2X purinoceptor 7
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: PALMITIC ACID
  • Ligand: water

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Supramolecule #1: Membrane protein

SupramoleculeName: Membrane protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus (rat)

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Macromolecule #1: P2X purinoceptor 7

MacromoleculeName: P2X purinoceptor 7 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Rattus (rat)
Molecular weightTheoretical: 68.472461 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPACCSWNDV FQYETNKVTR IQSVNYGTIK WILHMTVFSY VSFALMSDKL YQRKEPLISS VHTKVKGVAE VTENVTEGGV TKLVHGIFD TADYTLPLQG NSFFVMTNYL KSEGQEQKLC PEYPSRGKQC HSDQGCIKGW MDPQSKGIQT GRCIPYDQKR K TCEIFAWC ...String:
MPACCSWNDV FQYETNKVTR IQSVNYGTIK WILHMTVFSY VSFALMSDKL YQRKEPLISS VHTKVKGVAE VTENVTEGGV TKLVHGIFD TADYTLPLQG NSFFVMTNYL KSEGQEQKLC PEYPSRGKQC HSDQGCIKGW MDPQSKGIQT GRCIPYDQKR K TCEIFAWC PAEEGKEAPR PALLRSAENF TVLIKNNIDF PGHNYTTRNI LPGMNISCTF HKTWNPQCPI FRLGDIFQEI GE NFTEVAV QGGIMGIEIY WDCNLDSWSH RCQPKYSFRR LDDKYTNESL FPGYNFRYAK YYKENGMEKR TLIKAFGVRF DIL VFGTGG KFDIIQLVVY IGSTLSYFGL ATVCIDLIIN TYASTCCRSR VYPSCKCCEP CAVNEYYYRK KCEPIVEPKP TLKY VSFVD EPHIWMVDQQ LLGKSLQDVK GQEVPRPQTD FLELSRLSLS LHHSPPIPGQ PEEMQLLQIE AVPRSRDSPD WCQCG NCLP SQLPENRRAL EELCCRRKPG QCITTSELFS KIVLSREALQ LLLLYQEPLL ALEGEAINSK LRHCAYRSYA TWRFVS QDM ADFAILPSCC RWKIRKEFPK TQGQYSGFKY PY

UniProtKB: P2X purinoceptor 7

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Macromolecule #2: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / 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 #5: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 5 / Number of copies: 15 / Formula: PLM
Source (natural)Organism: Rattus (rat)
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 231 / 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

BufferpH: 7
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 11353 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6u9v

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 273076
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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