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- EMDB-13941: human Connexin 26 at 55mmHg PCO2, pH7.4: two masked subunits, class B -

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Basic information

Entry
Database: EMDB / ID: EMD-13941
Titlehuman Connexin 26 at 55mmHg PCO2, pH7.4: two masked subunits, class B
Map dataclass 2 dimer for human connexin26 in 55mmHg PCO2, pH7.4
Sample
  • Organelle or cellular component: two subunits masked from dodecameric assembly of human connexin 26: class B
    • 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 / gap junction channel activity involved in cell communication by electrical coupling / epididymis development / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction ...Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / epididymis development / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction / astrocyte projection / Gap junction assembly / gap junction channel activity / endoplasmic reticulum-Golgi intermediate compartment / inner ear development / decidualization / lateral plasma membrane / response to retinoic acid / cellular response to glucagon stimulus / cellular response to dexamethasone stimulus / response to ischemia / response to progesterone / sensory perception of sound / transmembrane transport / cell-cell signaling / response to estradiol / 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: 2.7 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) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateJun 15, 2022-
Current statusJun 15, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13941.png

Downloads & links

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Map

FileDownload / File: emd_13941.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationclass 2 dimer for human connexin26 in 55mmHg PCO2, pH7.4
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 184 pix.
= 200.56 Å		1.09 Å/pix.
x 184 pix.
= 200.56 Å		1.09 Å/pix.
x 184 pix.
= 200.56 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: 0.01
Minimum - Maximum-0.019027168 - 0.06631641
Average (Standard dev.)0.00016213318 (±0.0017110418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 200.56001 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : two subunits masked from dodecameric assembly of human connexin 2...

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

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Supramolecule #1: two subunits masked from dodecameric assembly of human connexin 2...

SupramoleculeName: two subunits masked from dodecameric assembly of human connexin 26: class B
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: 2 / 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: 4 / 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: 2 / 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: 7 / 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

Concentration3.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
100.0 mMNaClsodium 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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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: 1679668 / Details: LoG picker implemented in Relion3.1-beta
CTF correctionSoftware - Name: Gctf
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 36250
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2zw3

RefinementSpace: REAL
Output model

PDB-7qeu:
human Connexin 26 at 55mmHg PCO2, pH7.4: two masked subunits, class B

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