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- EMDB-1341: Three-dimensional structure of a human connexin26 gap junction ch... -

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Basic information

Entry
Database: EMDB / ID: EMD-1341
TitleThree-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule.
Map dataThree Dimensional Structure of a Human Connexin26 Gap Junction Channel
Sample
  • Sample: Human Connexin26 Gap Junction Channel
  • Protein or peptide: connexin26
Function / homologyGap junction beta-2 protein (Cx26) / connexin complex
Function and homology information
Biological speciesHomo sapiens (human)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 10.0 Å
AuthorsOshima A / Tani K / Hiroaki Y / Fujiyoshi Y / Sosinsky GE
CitationJournal: Proc Natl Acad Sci U S A / Year: 2007
Title: Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule.
Authors: Atsunori Oshima / Kazutoshi Tani / Yoko Hiroaki / Yoshinori Fujiyoshi / Gina E Sosinsky /
Abstract: Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of ...Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.
History
DepositionMar 12, 2007-
Header (metadata) releaseMar 19, 2007-
Map releaseJun 21, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.134696754
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.134696754
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1341.gif

Downloads & links

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Map

FileDownload / File: emd_1341.map.gz / Format: CCP4 / Size: 1.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThree Dimensional Structure of a Human Connexin26 Gap Junction Channel
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 151 pix.
= 302. Å		2.01 Å/pix.
x 57 pix.
= 114.407 Å		1.99 Å/pix.
x 57 pix.
= 113.186 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 1.98571 Å / Y: 2.00714 Å / Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.11 / Movie #1: 0.1346968
Minimum - Maximum-0.329032 - 0.434337
Average (Standard dev.)-0.0000118414 (±0.0612857)
SymmetrySpace group: 18
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-28-28-75
Dimensions5757151
Spacing5757151
CellA: 113.186 Å / B: 114.407 Å / C: 302 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.98571929824562.00714035087722
M x/y/z5757151
origin x/y/z0.0000.0000.000
length x/y/z113.186114.407302.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-28-28-75
NC/NR/NS5757151
D min/max/mean-0.3290.434-0.000

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Supplemental data

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Sample components

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Entire : Human Connexin26 Gap Junction Channel

EntireName: Human Connexin26 Gap Junction Channel
Components
  • Sample: Human Connexin26 Gap Junction Channel
  • Protein or peptide: connexin26

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Supramolecule #1000: Human Connexin26 Gap Junction Channel

SupramoleculeName: Human Connexin26 Gap Junction Channel / type: sample / ID: 1000 / Oligomeric state: 12 connexon form a gap junction / Number unique components: 1

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Macromolecule #1: connexin26

MacromoleculeName: connexin26 / type: protein_or_peptide / ID: 1 / Name.synonym: gap junction beta 2 / Number of copies: 12 / Oligomeric state: dodecamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cell membrane
Recombinant expressionOrganism: Sf9 cells / Recombinant plasmid: pBlueBac4.5
SequenceGO: connexin complex / InterPro: Gap junction beta-2 protein (Cx26)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration2 mg/mL
BufferpH: 5.8
Details: 10 mM MES, 100 mM NaCl, 50 mM MgCl2, 5 mM CaCl2, 2 mM DTT, 100 uM carbenoxolone (SIGMA), 0.005% NaN3, 1% glycerol
StainingType: NEGATIVE / Details: Vitrification
GridDetails: molybdenum grid covered with a thin carbon film
VitrificationCryogen name: NITROGEN / Chamber temperature: 100 K / Instrument: REICHERT-JUNG PLUNGER / Details: Vitrification instrument: Reichert plunger
Method: The grid was blotted with filter paper and plunged into liquid nitrogen.
Detailscrystals grown in dialysis buffer
Crystal formationDetails: crystals grown in dialysis buffer

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Electron microscopy

MicroscopeJEOL KYOTO-3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59100 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 19.4 µm / Nominal defocus min: 4.7 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Top entry liquid helium cooled cryo specimen holder.
Specimen holder model: OTHER / Tilt angle max: 45 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 45 °
TemperatureMin: 4.0 K / Max: 4.0 K / Average: 4.0 K
Alignment procedureLegacy - Astigmatism: objective astigmatism was corrected using a quadrupole stigmator at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 254 / Average electron dose: 25 e/Å2 / Bits/pixel: 12
Tilt angle min0

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Image processing

Crystal parametersUnit cell - A: 112.4 Å / Unit cell - B: 111.2 Å / Unit cell - C: 300.0 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 2 21 21
CTF correctionDetails: Each crystal
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Software - Name: MRC

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