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- PDB-8gts: CryoEM structure of human Pannexin1 with R217H congenital mutation. -

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Basic information

Entry
Database: PDB / ID: 8gts
TitleCryoEM structure of human Pannexin1 with R217H congenital mutation.
ComponentsPannexin-1
KeywordsMEMBRANE PROTEIN / Pannexin1 / ATP release / large-pore ion channel
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsHussain, N. / Penmatsa, A.
Funding support India, 1items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Jun 19, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pannexin-1
B: Pannexin-1
C: Pannexin-1
D: Pannexin-1
E: Pannexin-1
F: Pannexin-1
G: Pannexin-1


Theoretical massNumber of molelcules
Total (without water)336,5247
Polymers336,5247
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Pannexin-1


Mass: 48074.816 Da / Num. of mol.: 7 / Mutation: R217H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PANX1, MRS1, UNQ2529/PRO6028 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: Q96RD7

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Pannexin 1 (R217H) / Type: ORGANELLE OR CELLULAR COMPONENT
Details: human isoform 1 of Pannexin. Expressed in plasma membranes involved in ATP release
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 48 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S / Plasmid: pEG Bacmam
Buffer solutionpH: 8
Details: Fresh solution containing detergent was prepared for every prep.
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisTris1
2100 mMpotassium chlorideKCl1
31 %glycerolGlycerol1
450 microMGlycodiosgeninGDN1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample is homoheptamer purified to homogeneity.
Specimen supportDetails: 25 milli amps for 60s / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K
Details: sequentially blotted for 2 and 3.5 seconds with a wait of 10 s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 130000 X / Nominal defocus max: 3300 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K
Image recordingElectron dose: 41.12 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1776
EM imaging opticsEnergyfilter name: GIF Bioquantum / Chromatic aberration corrector: None / Details: Bioquantum with K2 camera / Energyfilter slit width: 20 eV / Phase plate: OTHER / Spherical aberration corrector: None
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selectionBlobpicker
2EPUimage acquisition
4cryoSPARCv3CTF correctionpatch CTF
7Coot9.03model fitting
11cryoSPARCclassification2D classification
12cryoSPARC3D reconstructionnon uniform refinement
13PHENIXmodel refinementreal space refine
Image processingDetails: Images were screened for ice thickness
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 781859 / Details: selected through particle picking
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40873 / Num. of class averages: 5 / Symmetry type: POINT
Atomic model buildingB value: 156 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coeficient
Atomic model buildingPDB-ID: 6WBF

6wbf


Pdb chain-ID: A / Accession code: 6WBF / Source name: PDB / Type: experimental model

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