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- EMDB-13938: human Connexin 26 dodecamer at 55mm Hg PCO2, pH7.4 -

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Basic information

Entry
Database: EMDB / ID: EMD-13938
Titlehuman Connexin 26 dodecamer at 55mm Hg PCO2, pH7.4
Map datamasked D6 .mrc file
Sample
  • Organelle or cellular component: dodecameric assembly of human connexin 26
    • Protein or peptide: Gap junction beta-2 protein
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water
Function / homology
Function and homology information


Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / epididymis development / 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 assembly ...Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / epididymis development / 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 assembly / astrocyte projection / gap junction channel activity / gap junction / cellular response to glucagon stimulus / inner ear development / decidualization / endoplasmic reticulum-Golgi intermediate compartment / lateral plasma membrane / response to retinoic acid / cellular response to dexamethasone stimulus / response to progesterone / response to ischemia / sensory perception of sound / transmembrane transport / response to estradiol / cell-cell signaling / cellular response to oxidative stress / cell body / response to lipopolysaccharide / calcium ion binding / perinuclear region of cytoplasm / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsBrotherton DH / Cameron AD / Savva CG / Ragan TJ
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P010393/1 United Kingdom
Leverhulme TrustRPG-2015-090 United Kingdom
CitationJournal: Structure / Year: 2022
Title: Conformational changes and CO-induced channel gating in connexin26.
Authors: Deborah H Brotherton / Christos G Savva / Timothy J Ragan / Nicholas Dale / Alexander D Cameron /
Abstract: Connexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. ...Connexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. Opening or closing of the channels is controlled by a variety of stimuli, and dysregulation leads to multiple diseases. An increase in the partial pressure of carbon dioxide (PCO) has been shown to cause connexin26 (Cx26) gap junctions to close. Here, we use cryoelectron microscopy (cryo-EM) to determine the structure of human Cx26 gap junctions under increasing levels of PCO. We show a correlation between the level of PCO and the size of the aperture of the pore, governed by the N-terminal helices that line the pore. This indicates that CO alone is sufficient to cause conformational changes in the protein. Analysis of the conformational states shows that movements at the N terminus are linked to both subunit rotation and flexing of the transmembrane helices.
History
DepositionDec 3, 2021-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateMay 18, 2022-
Current statusMay 18, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13938.png

Downloads & links

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Map

FileDownload / File: emd_13938.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmasked D6 .mrc file
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 340 pix.
= 370.6 Å		1.09 Å/pix.
x 340 pix.
= 370.6 Å		1.09 Å/pix.
x 340 pix.
= 370.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0115
Minimum - Maximum-0.025766028 - 0.09305251
Average (Standard dev.)0.00013552907 (±0.0018462121)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 370.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: human connexin 26 dodecamer at 55mmHg PCO2 half...

Fileemd_13938_half_map_1.map
Annotationhuman connexin 26 dodecamer at 55mmHg PCO2 half map 2 from full dataset
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: human connexin 26 dodecamer at 55mmHg PCO2 half...

Fileemd_13938_half_map_2.map
Annotationhuman connexin 26 dodecamer at 55mmHg PCO2 half map 1 from full dataset
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dodecameric assembly of human connexin 26

EntireName: dodecameric assembly of human connexin 26
Components
  • Organelle or cellular component: dodecameric assembly of human connexin 26
    • Protein or peptide: Gap junction beta-2 protein
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water

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Supramolecule #1: dodecameric assembly of human connexin 26

SupramoleculeName: dodecameric assembly of human connexin 26 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Gap junction beta-2 protein

MacromoleculeName: Gap junction beta-2 protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.713674 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP ISHIRLWALQ LIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQKVRIE GSLWWTYTSS IFFRVIFEAA FMYVFYVMYD G FSMQRLVK ...String:
MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP ISHIRLWALQ LIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQKVRIE GSLWWTYTSS IFFRVIFEAA FMYVFYVMYD G FSMQRLVK CNAWPCPNTV DCFVSRPTEK TVFTVFMIAV SGICILLNVT ELCYLLIRYC SGKSKKPVLV PR

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Macromolecule #2: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 2 / Number of copies: 48 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Macromolecule #3: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 3 / Number of copies: 12 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 305 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration3.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
5.0 %C3H8O3glycerol
1.0 mMC4H10O2S2DTT
0.03 %C24H46O11DDM
50.0 mMNaHCO3sodium hydrogen carbonate
1.25 mMNaH2PO4sodium dihydrogen phosphate
3.0 mMKClpotassium chloride
1.0 mMMgSO4magnesium sulphate
2.0 mMMgCl2magnesium chloride

Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of use. The basal buffer was filtered and de-gassed, and DDM and DTT added. The buffer was pH corrected at point of ...Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of use. The basal buffer was filtered and de-gassed, and DDM and DTT added. The buffer was pH corrected at point of use to 7.4 using 10% CO2 in 90% N2.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
Details: 3 microlitres protein applied to grid, blot time 6 seconds, in 10% CO2/90%N2 atmosphere.
DetailsThis sample monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4003 / Average exposure time: 5.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1679668 / Details: LoG picker implemented in Relion3.1-beta
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 344085
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2zw3

RefinementSpace: REAL
Output model

PDB-7qer:
human Connexin 26 dodecamer at 55mm Hg PCO2, pH7.4

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