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- EMDB-34265: CryoEM structure of human Pannexin isoform 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-34265
TitleCryoEM structure of human Pannexin isoform 3
Map dataMain Map of Pannexin 3
Sample
  • Organelle or cellular component: Pannexin 3
    • Protein or peptide: Pannexin-3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: PHOSPHATIDYLETHANOLAMINE
KeywordsPannexins / large pore ion-channels / MEMBRANE PROTEIN
Function / homology
Function and homology information


gap junction hemi-channel activity / wide pore channel activity / positive regulation of interleukin-1 production / gap junction / monoatomic cation transport / calcium channel activity / osteoblast differentiation / cell-cell signaling / endoplasmic reticulum membrane / structural molecule activity / 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: 3.91 Å
AuthorsHussain N / Penmatsa A
Funding support India, 1 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/15/2/502063 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_34265.png

Downloads & links

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Map

FileDownload / File: emd_34265.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain Map of Pannexin 3
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-1.618716 - 3.105998
Average (Standard dev.)0.011120179 (±0.06648128)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 383.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Panx3 halfmapB

Fileemd_34265_half_map_1.map
AnnotationPanx3_halfmapB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Panx3 halfmapA

Fileemd_34265_half_map_2.map
AnnotationPanx3_halfmapA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pannexin 3

EntireName: Pannexin 3
Components
  • Organelle or cellular component: Pannexin 3
    • Protein or peptide: Pannexin-3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: PHOSPHATIDYLETHANOLAMINE

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Supramolecule #1: Pannexin 3

SupramoleculeName: Pannexin 3 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: human isoform 3 of Pannexin. Expressed in ER and Plasma membranes involved in calcium and ATP regulation
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.68 kDa/nm

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Macromolecule #1: Pannexin-3

MacromoleculeName: Pannexin-3 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.734219 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSLAHTAAEY MLSDALLPDR RGPRLKGLRL ELPLDRIVKF VAVGSPLLLM SLAFAQEFSS GSPISCFSPS NFSIRQAAYV DSSCWDSLL HHKQDGPGQD KMKSLWPHKA LPYSLLALAL LMYLPVLLWQ YAAVPALSSD LLFIISELDK SYNRSIRLVQ H MLKIRQKS ...String:
MSLAHTAAEY MLSDALLPDR RGPRLKGLRL ELPLDRIVKF VAVGSPLLLM SLAFAQEFSS GSPISCFSPS NFSIRQAAYV DSSCWDSLL HHKQDGPGQD KMKSLWPHKA LPYSLLALAL LMYLPVLLWQ YAAVPALSSD LLFIISELDK SYNRSIRLVQ H MLKIRQKS SDPYVFWNEL EKARKERYFE FPLLERYLAC KQRSHSLVAT YLLRNSLLLI FTSATYLYLG HFHLDVFFQE EF SCSIKTG LLSDETHVPN LITCRLTSLS IFQIVSLSSV AIYTILVPVI IYNLTRLCRW DKRLLSVYEM LPAFDLLSRK MLG CPINDL NVILLFLRAN ISELISFSWL SVLCVLKDTT TQKHNIDTVV DFMTLLAGLE PSKPKHLTNS ACDEHP

UniProtKB: Pannexin-3

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 7 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 3 / Number of copies: 7 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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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

Concentration5 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. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV / Details: blotted for 3.5 seconds.
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: Bioquantum with K2 camera
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 1122 / Average electron dose: 48.8 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: 3.3000000000000003 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 130000
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: 411263
Startup modelType of model: OTHER / Details: AlphaFold2 model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: non uniform refinement / Number images used: 31517
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 34 / Avg.num./class: 926 / Software - Name: cryoSPARC / Software - details: 2D classification
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 143.6 / Target criteria: Correlation coeficient
Output model

PDB-8gtr:
CryoEM structure of human Pannexin isoform 3

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