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- EMDB-18670: Cryo-EM structure of Cx26 from Gallus Gallus in bicarbonate buffer -

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Basic information

Entry
Database: EMDB / ID: EMD-18670
TitleCryo-EM structure of Cx26 from Gallus Gallus in bicarbonate buffer
Map dataFull map D6 averaged
Sample
  • Complex: Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4
    • Protein or peptide: Gap junction protein
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water
KeywordsGap junction Large Pore Channel Carbon dioxide sensitive / MEMBRANE PROTEIN
Function / homology
Function and homology information


Gap junction assembly / gap junction-mediated intercellular transport / connexin complex / gap junction channel activity / sensory perception of sound / cell-cell signaling
Similarity search - Function
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 protein
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.07 Å
AuthorsBrotherton DH / Cameron AD
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: J Physiol / Year: 2025
Title: Multiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO.
Authors: Sarbjit Nijjar / Deborah Brotherton / Jack Butler / Valentin-Mihai Dospinescu / Harry G Gannon / Victoria Linthwaite / Martin Cann / Alexander Cameron / Nicholas Dale /
Abstract: CO directly modifies the gating of connexin26 (Cx26) gap junction channels and hemichannels. This gating depends upon Lys125, and the proposed mechanism involves carbamylation of Lys125 to allow ...CO directly modifies the gating of connexin26 (Cx26) gap junction channels and hemichannels. This gating depends upon Lys125, and the proposed mechanism involves carbamylation of Lys125 to allow formation of a salt bridge with Arg104 on the neighbouring subunit. We demonstrate via carbamate trapping and tandem mass spectrometry that five Lys residues within the cytoplasmic loop, including Lys125, are indeed carbamylated by CO. The cytoplasmic loop appears to provide a chemical microenvironment that facilitates carbamylation. Systematic mutation of these Lys residues to Arg shows that only carbamylation of Lys125 is essential for hemichannel opening. By contrast, carbamylation of Lys108 and Lys125 is essential for gap junction closure to CO. Chicken (Gallus gallus) Cx26 gap junction channels lack Lys108 and do not close to CO, as shown by both a dye transfer assay and a high-resolution cryogenic electron microscopy structure. The mutation Lys108Arg prevents CO-dependent gap junction channel closure in human Cx26. Our findings directly demonstrate carbamylation in connexins, provide further insight into the differential action of CO on Cx26 hemichannels and gap junction channels, and increase support for the role of the N-terminus in gating the Cx26 channel. KEY POINTS: Direct evidence of carbamylation of multiple lysine residues in the cytoplasmic loop of Cx26. Concentration-dependent carbamylation at lysines 108, 122 and 125. Only carbamylation of lysine 125 is essential for hemichannel opening to CO. Carbamylation of lysine 108 along with lysine 125 is essential for CO-dependent gap junction channel closure.
History
DepositionOct 18, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18670.png

Downloads & links

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Map

FileDownload / File: emd_18670.map.gz / Format: CCP4 / Size: 316.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map D6 averaged
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 436 pix.
= 370.6 Å		0.85 Å/pix.
x 436 pix.
= 370.6 Å		0.85 Å/pix.
x 436 pix.
= 370.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0075
Minimum - Maximum-0.0151488595 - 0.054582115
Average (Standard dev.)0.000016091963 (±0.0013317354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions436436436
Spacing436436436
CellA=B=C: 370.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map D6 averaged

Fileemd_18670_half_map_1.map
AnnotationHalf map D6 averaged
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map D6 averaged

Fileemd_18670_half_map_2.map
AnnotationHalf map D6 averaged
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4

EntireName: Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4
Components
  • Complex: Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4
    • Protein or peptide: Gap junction protein
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water

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Supramolecule #1: Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4

SupramoleculeName: Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Gap junction protein

MacromoleculeName: Gap junction protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 26.264119 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDWGTLQAVL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA ERVWGDEQQD FVCNTLQPGC RNVCYDHFFP ISHIRLWALQ LIFVSTPAL LVAMHVAYTR HERKRRFRSG DKINIEELKN EKIHIRGPLW WTYTCSIFFR IVFEAVFMYV FYYMYDGYQM P RLVKCDAW ...String:
MDWGTLQAVL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA ERVWGDEQQD FVCNTLQPGC RNVCYDHFFP ISHIRLWALQ LIFVSTPAL LVAMHVAYTR HERKRRFRSG DKINIEELKN EKIHIRGPLW WTYTCSIFFR IVFEAVFMYV FYYMYDGYQM P RLVKCDAW PCPNVVDCFV SRPTEKTTFT IFMLAVSGIC MMLNLAELCY LVIKVCLKDS GKTTVLK

UniProtKB: Gap junction protein

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Macromolecule #2: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 2 / Number of copies: 24 / 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: 24 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 466 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
70.0 mMNaClsodium chloride
5.0 %C3H8O3glycerol
1.0 mMC4H10O2S2DTT
0.03 %C2H46O11DDM
80.0 mMNaH2P04sodium hydorgen carbonate
3.0 mMKClpotassium chloride
1.0 mMMgSO4magnesium sulphate
4.0 mMMgCl2magnesium chloride

Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of used. The basal buffer was filtered and degassed 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 used. The basal buffer was filtered and degassed and DDM and DTT added. The buffer was pH corrected at point of use to pH 7.4 using 15% CO2 in 85% N2.
VitrificationCryogen name: ETHANE-PROPANE / Instrument: LEICA EM GP
Details: 3 microlitres protein applied to grid, blot time 7 seconds, in 15% CO2/85%N2 atmosphere.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 8713 / Average exposure time: 3.0 sec. / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2041681
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.07 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Software - details: beta / Number images used: 161947
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 6
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7qeq


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-8qvn:
Cryo-EM structure of Cx26 from Gallus Gallus in bicarbonate buffer

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