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- EMDB-21593: Cryo-EM structure of human Pannexin 1 channel N255A mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-21593
TitleCryo-EM structure of human Pannexin 1 channel N255A mutant
Map dataUnsharpened map of N255A-hsPANX1-Hemi
Sample
  • Complex: Wild type human Pannexin 1 channel
    • Protein or peptide: Pannexin-1
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
  • Ligand: DIACYL GLYCEROLDiglyceride
  • Ligand: CHOLESTEROL
Function / homology
Function and homology information


Electric Transmission Across Gap Junctions / leak channel activity / positive regulation of interleukin-1 alpha production / bleb / wide pore channel activity / gap junction channel activity / gap junction / positive regulation of macrophage cytokine production / oogenesis / response to ATP ...Electric Transmission Across Gap Junctions / leak channel activity / positive regulation of interleukin-1 alpha production / bleb / wide pore channel activity / gap junction channel activity / gap junction / positive regulation of macrophage cytokine production / oogenesis / response to ATP / monoatomic cation transport / The NLRP3 inflammasome / positive regulation of interleukin-1 beta production / response to ischemia / calcium channel activity / calcium ion transport / actin filament binding / cell-cell signaling / scaffold protein binding / protease binding / transmembrane transporter binding / signaling receptor binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
Pannexin / Innexin / Innexin / Pannexin family profile.
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsLu W / Du J / Ruan Z
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R56HL144929 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS111031 United States
American Heart Association20POST35120556 United States
CitationJournal: Nature / Year: 2020
Title: Structures of human pannexin 1 reveal ion pathways and mechanism of gating.
Authors: Zheng Ruan / Ian J Orozco / Juan Du / Wei Lü /
Abstract: Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation, apoptotic cell clearance and human oocyte development. ...Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation, apoptotic cell clearance and human oocyte development. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
History
DepositionMar 26, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateJun 30, 2021-
Current statusJun 30, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0075
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0075
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wbm
  • Surface level: 0.0075
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21593.png

Downloads & links

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Map

FileDownload / File: emd_21593.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map of N255A-hsPANX1-Hemi
Voxel sizeX=Y=Z: 0.812 Å
Density
Contour LevelBy AUTHOR: 0.0075 / Movie #1: 0.0075
Minimum - Maximum-0.008942808 - 0.033615544
Average (Standard dev.)0.00010615531 (±0.0018125285)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 243.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8120.8120.812
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z243.600243.600243.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0090.0340.000

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Supplemental data

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Additional map: Sharpened map of N255A-hsPANX1-Hemi

Fileemd_21593_additional.map
AnnotationSharpened map of N255A-hsPANX1-Hemi
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Wild type human Pannexin 1 channel

EntireName: Wild type human Pannexin 1 channel
Components
  • Complex: Wild type human Pannexin 1 channel
    • Protein or peptide: Pannexin-1
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
  • Ligand: DIACYL GLYCEROLDiglyceride
  • Ligand: CHOLESTEROL

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Supramolecule #1: Wild type human Pannexin 1 channel

SupramoleculeName: Wild type human Pannexin 1 channel / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Pannexin-1

MacromoleculeName: Pannexin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.204418 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAIAQLATEY VFSDFLLKEP TEPKFKGLRL ELAVDKMVTC IAVGLPLLLI SLAFAQEISI GTQISCFSPS SFSWRQAAFV DSYCWAAVQ QKNSLQSESG NLPLWLHKFF PYILLLFAIL LYLPPLFWRF AAAPHICSDL KFIMEELDKV YNRAIKAAKS A RDLDMRDG ...String:
MAIAQLATEY VFSDFLLKEP TEPKFKGLRL ELAVDKMVTC IAVGLPLLLI SLAFAQEISI GTQISCFSPS SFSWRQAAFV DSYCWAAVQ QKNSLQSESG NLPLWLHKFF PYILLLFAIL LYLPPLFWRF AAAPHICSDL KFIMEELDKV YNRAIKAAKS A RDLDMRDG ACSVPGVTEN LGQSLWEVSE SHFKYPIVEQ YLKTKKNSNN LIIKYISCRL LTLIIILLAC IYLGYYFSLS SL SDEFVCS IKSGILRADS TVPDQFQCKL IAVGIFQLLS VINLVVYVLL APVVVYTLFV PFRQKTDVLK VYEILPTFDV LHF KSEGYN DLSLYNLFLE ENISEVKSYK CLKVLENIKS SGQGIDPMLL LTNLGMI

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Macromolecule #2: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 2 / Number of copies: 14 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #3: 1,2-Distearoyl-sn-glycerophosphoethanolamine

MacromoleculeName: 1,2-Distearoyl-sn-glycerophosphoethanolamine / type: ligand / ID: 3 / Number of copies: 7 / Formula: 3PE
Molecular weightTheoretical: 748.065 Da
Chemical component information

ChemComp-3PE:
1,2-Distearoyl-sn-glycerophosphoethanolamine / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #4: DIACYL GLYCEROL

MacromoleculeName: DIACYL GLYCEROL / type: ligand / ID: 4 / Number of copies: 7 / Formula: DGA
Molecular weightTheoretical: 625.018 Da
Chemical component information

ChemComp-DGA:
DIACYL GLYCEROL / Diglyceride

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 14 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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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

Concentration7 mg/mL
BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291.15 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 8.0 sec. / Average electron dose: 49.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3945051
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: cryoSPARC ab-initio reconstruction
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1b)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1b)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1b)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1b) / Number images used: 407341

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6wbm:
Cryo-EM structure of human Pannexin 1 channel N255A mutant

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