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- EMDB-34267: Cryo-EM structure of human Pannexin1 resembling Pannexin2 pore wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-34267
TitleCryo-EM structure of human Pannexin1 resembling Pannexin2 pore with W74R/R75Dmutations
Map datamain map for coordinates
Sample
  • Complex: Pannexin 1 (W74R/R75D)
    • Protein or peptide: Pannexin-1
KeywordsPannexin1 / ATP release / large-pore ion channel. / MEMBRANE PROTEIN
Function / homology
Function and homology information


ATP transmembrane transporter activity / ATP transport / leak channel activity / Electric Transmission Across Gap Junctions / positive regulation of interleukin-1 alpha production / wide pore channel activity / bleb / monoatomic anion channel activity / monoatomic anion transmembrane transport / gap junction ...ATP transmembrane transporter activity / ATP transport / leak channel activity / Electric Transmission Across Gap Junctions / positive regulation of interleukin-1 alpha production / wide pore channel activity / bleb / monoatomic anion channel activity / monoatomic anion transmembrane transport / gap junction / gap junction channel activity / positive regulation of macrophage cytokine production / response to ATP / oogenesis / 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 / identical protein binding / membrane / 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: 3.2 Å
AuthorsHussain N / Penmatsa A
Funding support India, 1 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/S/22/1/506242 India
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures of pannexin 1 and 3 reveal differences among pannexin isoforms.
Authors: Nazia Hussain / Ashish Apotikar / Shabareesh Pidathala / Sourajit Mukherjee / Ananth Prasad Burada / Sujit Kumar Sikdar / Kutti R Vinothkumar / Aravind Penmatsa /
Abstract: Pannexins are single-membrane large-pore channels that release ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining ...Pannexins are single-membrane large-pore channels that release ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining residues. In this study, we report the cryo-EM structure of pannexin 3 at 3.9 Å and analyze its structural differences with pannexin isoforms 1 and 2. The pannexin 3 vestibule has two distinct chambers and a wider pore radius in comparison to pannexins 1 and 2. We further report two cryo-EM structures of pannexin 1, with pore substitutions W74R/R75D that mimic the pore lining residues of pannexin 2 and a germline mutant of pannexin 1, R217H at resolutions of 3.2 Å and 3.9 Å, respectively. Substitution of cationic residues in the vestibule of pannexin 1 results in reduced ATP interaction propensities to the channel. The germline mutant R217H in transmembrane helix 3 (TM3), leads to a partially constricted pore, reduced ATP interaction and weakened voltage sensitivity. The study compares the three pannexin isoform structures, the effects of substitutions of pore and vestibule-lining residues and allosteric effects of a pathological substitution on channel structure and function thereby enhancing our understanding of this vital group of ATP-release channels.
History
DepositionSep 8, 2022-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34267.png

Downloads & links

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Map

FileDownload / File: emd_34267.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map for coordinates
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 302.4 Å		0.84 Å/pix.
x 360 pix.
= 302.4 Å		0.84 Å/pix.
x 360 pix.
= 302.4 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.9583318 - 1.527624
Average (Standard dev.)0.00041860502 (±0.030946907)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map partA

Fileemd_34267_half_map_1.map
Annotationhalf map partA
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half map part B

Fileemd_34267_half_map_2.map
Annotationhalf map part B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pannexin 1 (W74R/R75D)

EntireName: Pannexin 1 (W74R/R75D)
Components
  • Complex: Pannexin 1 (W74R/R75D)
    • Protein or peptide: Pannexin-1

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Supramolecule #1: Pannexin 1 (W74R/R75D)

SupramoleculeName: Pannexin 1 (W74R/R75D) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: human isoform 1 of Pannexin. Expressed in plasma membranes involved in ATP release
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48 kDa/nm

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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: 49.127902 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAIAQLATEY VFSDFLLKEP TEPKFKGLRL ELAVDKMVTC IAVGLPLLLI SLAFAQEISI GTQISCFSPS SFSRDQAAFV DSYCWAAVQ QKNSLQSESG NLPLWLHKFF PYILLLFAIL LYLPPLFWRF AAAPHICSDL KFIMEELDKV YNRAIKAAKS A RDLDMRDG ...String:
MAIAQLATEY VFSDFLLKEP TEPKFKGLRL ELAVDKMVTC IAVGLPLLLI SLAFAQEISI GTQISCFSPS SFSRDQAAFV DSYCWAAVQ QKNSLQSESG NLPLWLHKFF PYILLLFAIL LYLPPLFWRF AAAPHICSDL KFIMEELDKV YNRAIKAAKS A RDLDMRDG ACSVPGVTEN LGQSLWEVSE SHFKYPIVEQ YLKTKKNSNN LIIKYISCRL LTLIIILLAC IYLGYYFSLS SL SDEFVCS IKSGILRNDS TVPDQFQCKL IAVGIFQLLS VINLVVYVLL APVVVYTLFV PFRQKTDVLK VYEILPTFDV LHF KSEGYN DLSLYNLFLE ENISEVKSYK CLKVLENIKS SGQGIDPMLL LTNLGMIKMD VVDGKTPMSA EMREEQGNQT AELQ GMNID SETKANNGEK NARQRLLDSS CHHHHHHHH

UniProtKB: Pannexin-1

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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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMTrisTris
100.0 mMKClpotassium chloride
1.0 %Glycerolglycerol
50.0 microMGDNGlycodiosgenin

Details: Fresh solution containing detergent was prepared for every prep.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV / Details: 3.5 s single blot.
DetailsSample is homoheptamer purified to homogeneity.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K / Max: 100.0 K
Specialist opticsPhase plate: OTHER / Spherical aberration corrector: None / Chromatic aberration corrector: None / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV / Details: Gatan k3
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 15550 / Average electron dose: 39.84 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 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

DetailsImages were screened for ice thickness
Particle selectionNumber selected: 3869579 / Details: selected through particle picking
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6wbf

Final reconstructionNumber classes used: 23 / Applied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: non uniform refinement / Number images used: 80029
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 23 / Avg.num./class: 15000 / Software - Name: cryoSPARC / Software - details: 2D classification
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6wbf


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 106.6 / Target criteria: Correlation coeficient
Output model

PDB-8gtt:
Cryo-EM structure of human Pannexin1 resembling Pannexin2 pore with W74R/R75Dmutations

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