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- PDB-8a3b: Cryo-EM structure of mouse Pannexin 1 purified in Salipro nanopar... -

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Basic information

Entry
Database: PDB / ID: 8a3b
TitleCryo-EM structure of mouse Pannexin 1 purified in Salipro nanoparticles
ComponentsPannexin-1
KeywordsMEMBRANE PROTEIN / Membrane transporter / ATP-release channel / inflammation / immune function
Function / homology
Function and homology information


Electric Transmission Across Gap Junctions / The NLRP3 inflammasome / 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 / response to ATP ...Electric Transmission Across Gap Junctions / The NLRP3 inflammasome / 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 / response to ATP / monoatomic cation transport / positive regulation of interleukin-1 beta production / response to ischemia / calcium channel activity / calcium ion transport / actin filament binding / cell-cell signaling / actin binding / scaffold protein binding / protease binding / transmembrane transporter binding / signaling receptor binding / protein-containing complex binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / protein-containing complex / identical protein binding / plasma membrane
Similarity search - Function
Pannexin / Innexin / Innexin / Pannexin family profile.
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDrulyte, I.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Sci Rep / Year: 2023
Title: Direct cell extraction of membrane proteins for structure-function analysis.
Authors: Ieva Drulyte / Aspen Rene Gutgsell / Pilar Lloris-Garcerá / Michael Liss / Stefan Geschwindner / Mazdak Radjainia / Jens Frauenfeld / Robin Löving /
Abstract: Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach ...Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach for the incorporation of membrane proteins directly from cells into lipid Salipro nanoparticles. Here, with the pannexin1 channel as a case study, we demonstrate the applicability of this method for structure-function analysis using SPR and cryo-EM.
History
DepositionJun 7, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release

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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)353,3287
Polymers353,3287
Non-polymers00
Water0
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: 50475.434 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Panx1 / Production host: Homo sapiens (human) / Strain (production host): Expi293 / References: UniProt: Q9JIP4

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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: Salipro-mPANX1 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.353 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: Expi293
Buffer solutionpH: 7.5 / Details: 50 mM HEPES at pH 7.5, 150 mM NaCl
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow-discharged using 20 mAmp current for 45 sec and charge set to positive.
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K
Details: To overcome preferred orientation, 0.5 mM fluorinated Fos-Choline 8 was added to the sample just before the grid freezing. Blot parameter: blot force +20, blot time 10 s, waiting time 30s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Titan Krios G4 was used with fringe-free imaging and aberration-free image shifts. Nominal pixel size for 165kx 0.75 A, calibrated pixel size 0.727 A.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.16 sec. / Electron dose: 40.24 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7955
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2.Ximage acquisition
4CTFFIND4.1CTF correction
7UCSF ChimeraX1.3model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12REFMAC5.83D reconstruction
13ISOLDE1.3model refinement
14REFMAC5.8model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1314251
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 268823 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL

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