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- EMDB-1749: A structure of Cx26M34Adel2-7 at 10 angstrom resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-1749
TitleA structure of Cx26M34Adel2-7 at 10 angstrom resolution
Map dataThis is a three dimensional map of Cx26M34Adel2-7 at 10 angstrom resolution.
Sample
  • Sample: Human connexin26
  • Organelle or cellular component: Gap junction
Function / homology
Function and homology information


Transport of connexons to the plasma membrane / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction / gap junction channel activity / Gap junction assembly ...Transport of connexons to the plasma membrane / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction / gap junction channel activity / Gap junction assembly / endoplasmic reticulum-Golgi intermediate compartment / sensory perception of sound / transmembrane transport / cell-cell signaling / calcium ion binding / identical protein binding / plasma membrane
Similarity search - Function
Gap junction beta-2 protein (Cx26) / Connexin / Connexin, N-terminal / Connexin, conserved site / Gap junction protein, cysteine-rich domain / Connexin, N-terminal domain superfamily / Connexin / Connexins signature 1. / Connexins signature 2. / Connexin homologues / Gap junction channel protein cysteine-rich domain
Similarity search - Domain/homology
Gap junction beta-2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 10.0 Å
AuthorsOshima A / Tani K / Toloue MM / Hiroaki Y / Smock A / Inukai S / Cone A / Nicholson BJ / Sosinsky GE / Fujiyoshi Y
CitationJournal: J Mol Biol / Year: 2011
Title: Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.
Authors: Atsunori Oshima / Kazutoshi Tani / Masoud M Toloue / Yoko Hiroaki / Amy Smock / Sayaka Inukai / Angela Cone / Bruce J Nicholson / Gina E Sosinsky / Yoshinori Fujiyoshi /
Abstract: Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we ...Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration.
History
DepositionJun 11, 2010-
Header (metadata) releaseSep 2, 2010-
Map releaseJan 7, 2011-
UpdateOct 2, 2013-
Current statusOct 2, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iz2
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iz2
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3iz2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1749.png

Downloads & links

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Map

FileDownload / File: emd_1749.map.gz / Format: CCP4 / Size: 1.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a three dimensional map of Cx26M34Adel2-7 at 10 angstrom resolution.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 151 pix.
= 450. Å		2 Å/pix.
x 57 pix.
= 112. Å		2.03 Å/pix.
x 57 pix.
= 113.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 2.03 Å / Y: 2 Å / Z: 3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0015 / Movie #1: 0.0015
Minimum - Maximum-0.00961921 - 0.00998023
Average (Standard dev.)-0.00000374 (±0.00153846)
SymmetrySpace group: 18
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-28-28-75
Dimensions5757151
Spacing5656150
CellA: 113.68 Å / B: 112.0 Å / C: 450.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.0323
M x/y/z5656150
origin x/y/z0.0000.0000.000
length x/y/z113.680112.000450.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-100-100-100
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-28-28-75
NC/NR/NS5757151
D min/max/mean-0.0100.010-0.000

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Supplemental data

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Segmentation: This is a three dimensional map of Cx26M34Adel2-7...

AnnotationThis is a three dimensional map of Cx26M34Adel2-7 at 10 angstrom resolution
Fileemd_1749_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human connexin26

EntireName: Human connexin26
Components
  • Sample: Human connexin26
  • Organelle or cellular component: Gap junction

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Supramolecule #1000: Human connexin26

SupramoleculeName: Human connexin26 / type: sample / ID: 1000 / Oligomeric state: Dodecameric / Number unique components: 1
Molecular weightTheoretical: 300 KDa

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Supramolecule #1: Gap junction

SupramoleculeName: Gap junction / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Connexin / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pBlueBac4.5

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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, pH 5.8, 100 mM NaCl, 50 mM MgCl2, 5 mM CaCl2, 2 mM DTT, 100 uM carbenoxolone, 0.005% NaN3, 1% glycerol.
StainingType: NEGATIVE / Details: Embedded in ice with 10% trehalose
GridDetails: Molybdenum grid
VitrificationCryogen name: NITROGEN / Chamber temperature: 100 K / Instrument: LEICA KF80 / Details: Vitrification instrument: Reichert KF-80
Method: The grids were blotted with filter paper and fast frozen into liquid nitrogen
DetailsCrystals grown in three lipid bilayers
Crystal formationDetails: Crystals grown in three lipid bilayers

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Electron microscopy

MicroscopeJEOL KYOTO-3000SFF
TemperatureMin: 4 K / Max: 4 K / Average: 4 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected using a quadrupole stigmator at 250,000 magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 187 / Average electron dose: 25 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.6 mm / Nominal defocus max: 2.213 µm / Nominal defocus min: 0.544 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Top entry helium cooled cryo stage / Specimen holder model: JEOL / Tilt angle max: 45 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 45 °

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: OTHER / Software - Name: MRC
Crystal parametersUnit cell - A: 113.4 Å / Unit cell - B: 112.2 Å / Unit cell - C: 300 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Plane group: P 2 21 21
CTF correctionDetails: Each image

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Atomic model buiding 1

Initial modelPDB ID:

2zw3


Chain - Chain ID: A
SoftwareName: Situs
DetailsPDBEntryID_givenInChain. Protocol: Rigid body. The chain containing the amino acids from 18 to 217 corresponding to a connexin monomer was initially fitted manually into each subunit in the cryo-EM structure using program O.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Linear cross correlation
Output model

PDB-3iz2:
C-alpha model fitted into the EM structure of Cx26M34Adel2-7

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