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- EMDB-15110: Cryo-EM structure of mouse Pannexin 1 purified in Salipro nanopar... -

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Basic information

Entry
Database: EMDB / ID: EMD-15110
TitleCryo-EM structure of mouse Pannexin 1 purified in Salipro nanoparticles
Map dataDeepEMhancer sharpened map
Sample
  • Organelle or cellular component: Salipro-mPANX1
    • Protein or peptide: Pannexin-1
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDrulyte I
Funding support1 items
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, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15110.png

Downloads & links

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Map

FileDownload / File: emd_15110.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 256 pix.
= 279.168 Å		1.09 Å/pix.
x 256 pix.
= 279.168 Å		1.09 Å/pix.
x 256 pix.
= 279.168 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0905 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.0026665519 - 1.6870908
Average (Standard dev.)0.001648256 (±0.029222734)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 279.168 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_15110_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_15110_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_15110_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Salipro-mPANX1

EntireName: Salipro-mPANX1
Components
  • Organelle or cellular component: Salipro-mPANX1
    • Protein or peptide: Pannexin-1

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Supramolecule #1: Salipro-mPANX1

SupramoleculeName: Salipro-mPANX1 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 353 KDa

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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: Mus musculus (house mouse)
Molecular weightTheoretical: 50.475434 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSDYKDDDDK GGGGSMAIAH LATEYVFSDF LLKEPTEPKF KGLRLELAVD KMVTCIAVGL PLLLISLAFA QEISIGTQIS CFSPSSFSW RQAAFVDSYC WAAVQQKSSL QSESGNLPLW LHKFFPYILL LFAILLYLPA LFWRFSAAPH LCSDLKFIME E LDKVYNRA ...String:
MSDYKDDDDK GGGGSMAIAH LATEYVFSDF LLKEPTEPKF KGLRLELAVD KMVTCIAVGL PLLLISLAFA QEISIGTQIS CFSPSSFSW RQAAFVDSYC WAAVQQKSSL QSESGNLPLW LHKFFPYILL LFAILLYLPA LFWRFSAAPH LCSDLKFIME E LDKVYNRA IKAAKSARDL DLRDGPGPPG VTENVGQSLW EISESHFKYP IVEQYLKTKK NSSHLIMKYI SCRLVTFVVI LL ACIYLSY YFSLSSLSDE FLCSIKSGVL KNDSTIPDRF QCKLIAVGIF QLLSLINLIV YALLIPVVVY TFFIPFRQKT DIL KVYEIL PTFDVLHFKS EGYNDLSLYN LFLEENISEL KSYKCLKVLE NIKSNGQGID PMLLLTNLGM IKMDIIDGKI PTSL QTKGE DQGSQRVEFK DLDLSSEAAA NNGEKNSRQR LLNPSCLEVL FQ

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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

Concentration2.5 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES at pH 7.5, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
Details: Grids were glow-discharged using 20 mAmp current for 45 sec and charge set to positive.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsTitan 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.
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 7955 / Average exposure time: 4.16 sec. / Average electron dose: 40.24 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1314251
Startup modelType of model: INSILICO MODEL
Details: Ab Initio starting model was generated using cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Details: 3D classification with no particle alignment was used to select the best subset of particles.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: REFMAC (ver. 5.8) / Number images used: 268823
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Output model

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

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