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- EMDB-45177: Cryo-EM structure of the full-length human P2X4 receptor in the A... -

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Basic information

Entry
Database: EMDB / ID: EMD-45177
TitleCryo-EM structure of the full-length human P2X4 receptor in the ATP-bound desensitized state
Map dataLocally sharpened map for the human P2X4 receptor in the ATP-bound desensitized state
Sample
  • Complex: Membrane protein
    • Protein or peptide: P2X purinoceptor 4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: water
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 / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / purinergic nucleotide receptor signaling pathway / negative regulation of cardiac muscle hypertrophy / Elevation of cytosolic Ca2+ levels / ligand-gated calcium channel activity / response to fluid shear stress ...Platelet homeostasis / sensory perception of touch / positive regulation of microglial cell migration / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / purinergic nucleotide receptor signaling pathway / negative regulation of cardiac muscle hypertrophy / Elevation of cytosolic Ca2+ levels / ligand-gated calcium channel activity / response to fluid shear stress / positive regulation of prostaglandin secretion / regulation of chemotaxis / tissue homeostasis / positive regulation of calcium ion transport / endothelial cell activation / relaxation of cardiac muscle / positive regulation of endothelial cell chemotaxis / cellular response to zinc ion / cellular response to ATP / response to ATP / positive regulation of calcium ion transport into cytosol / parallel fiber to Purkinje cell synapse / behavioral response to pain / membrane depolarization / regulation of cardiac muscle contraction / positive regulation of blood vessel endothelial cell migration / response to axon injury / 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 / regulation of blood pressure / calcium ion transmembrane transport / 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 / lysosomal membrane / signaling receptor binding / 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
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsShi H / Ditter IA / Oken AC / 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: 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, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45177.png

Downloads & links

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Map

FileDownload / File: emd_45177.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally sharpened map for the human P2X4 receptor in the ATP-bound desensitized state
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: 0.05
Minimum - Maximum-0.0028888816 - 0.6203105
Average (Standard dev.)0.00014269822 (±0.0050582485)
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_45177_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map for the human P2X4 receptor in...

Fileemd_45177_additional_1.map
AnnotationUnsharpened map for the human P2X4 receptor in the ATP-bound desensitized state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map for the human P2X4 receptor in...

Fileemd_45177_additional_2.map
AnnotationSharpened map for the human P2X4 receptor in the ATP-bound desensitized state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A for the human P2X4 receptor...

Fileemd_45177_half_map_1.map
AnnotationHalf map A for the human P2X4 receptor in the ATP-bound desensitized state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B for the human P2X4 receptor...

Fileemd_45177_half_map_2.map
AnnotationHalf map B for the human P2X4 receptor in the ATP-bound desensitized state
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 4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • 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: Homo sapiens (human)

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Macromolecule #1: P2X purinoceptor 4

MacromoleculeName: P2X purinoceptor 4 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.415 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAGCCAALAA FLFEYDTPRI VLIRSRKVGL MNRAVQLLIL AYVIGWVFVW EKGYQETDSV VSSVTTKVKG VAVTNTSKLG FRIWDVADY VIPAQEENSL FVMTNVILTM NQTQGLCPEI PDATTVCKSD ASCTAGSAGT HSNGVSTGRC VAFNGSVKTC E VAAWCPVE ...String:
MAGCCAALAA FLFEYDTPRI VLIRSRKVGL MNRAVQLLIL AYVIGWVFVW EKGYQETDSV VSSVTTKVKG VAVTNTSKLG FRIWDVADY VIPAQEENSL FVMTNVILTM NQTQGLCPEI PDATTVCKSD ASCTAGSAGT HSNGVSTGRC VAFNGSVKTC E VAAWCPVE DDTHVPQPAF LKAAENFTLL VKNNIWYPKF NFSKRNILPN ITTTYLKSCI YDAKTDPFCP IFRLGKIVEN AG HSFQDMA VEGGIMGIQV NWDCNLDRAA SLCLPRYSFR RLDTRDVEHN VSPGYNFRFA KYYRDLAGNE QRTLIKAYGI RFD IIVFGK AGKFDIIPTM INIGSGLALL GMATVLCDII VLYCMKKRLY YREKKYKYVE DYEQGLASEL DQ

UniProtKB: P2X purinoceptor 4

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / 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 #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / 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 #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 249 / 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

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 15582 / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.9 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:

5svl

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 122405
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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