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- EMDB-18293: Cryo-EM structure of Cx26 solubilised in LMNG: classification on ... -

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Basic information

Entry
Database: EMDB / ID: EMD-18293
TitleCryo-EM structure of Cx26 solubilised in LMNG: classification on subunit A; Nconst-mon conformation
Map dataFull map locally sharpened in phenix; classification on A and masked refinement over A-F hemichannel
Sample
  • Complex: Connexin 26, 90mmHg PCO2, pH7.4, solubilised in LMNG.
    • Protein or peptide: Gap junction beta-2 protein
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water
KeywordsGap junction Large Pore Channel Carbon dioxide sensitive / MEMBRANE PROTEIN
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 / gap junction / astrocyte projection / gap junction channel activity / 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 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: 2.3 Å
AuthorsBrotherton DH / Savva CG / 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
Citation
Journal: Elife / Year: 2024
Title: Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO.
Authors: Deborah H Brotherton / Sarbjit Nijjar / Christos G Savva / Nicholas Dale / Alexander David Cameron /
Abstract: Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO. This is proposed to be mediated ...Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.
#1: Journal: Elife / Year: 2024
Title: Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2
Authors: Brotherton DH / Nijjar S / Savva CG / Dale N / Cameron AD
History
DepositionAug 22, 2023-
Header (metadata) releaseJun 12, 2024-
Map releaseJun 12, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18293.png

Downloads & links

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Map

FileDownload / File: emd_18293.map.gz / Format: CCP4 / Size: 316.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map locally sharpened in phenix; classification on A and masked refinement over A-F hemichannel
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.021978496 - 0.04424442
Average (Standard dev.)-0.000020879857 (±0.0008001975)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions436436436
Spacing436436436
CellA=B=C: 364.06 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18293_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map (unsharpened); classification on A and masked...

Fileemd_18293_additional_1.map
AnnotationFull map (unsharpened); classification on A and masked refinement over A-F hemichannel
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map (unsharpened); classification on A and masked...

Fileemd_18293_half_map_1.map
AnnotationHalf map (unsharpened); classification on A and masked refinement over A-F hemichannel
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map (unsharpened); classification on A and masked...

Fileemd_18293_half_map_2.map
AnnotationHalf map (unsharpened); classification on A and masked refinement over A-F hemichannel
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, 90mmHg PCO2, pH7.4, solubilised in LMNG.

EntireName: Connexin 26, 90mmHg PCO2, pH7.4, solubilised in LMNG.
Components
  • Complex: Connexin 26, 90mmHg PCO2, pH7.4, solubilised in LMNG.
    • Protein or peptide: Gap junction beta-2 protein
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water

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Supramolecule #1: Connexin 26, 90mmHg PCO2, pH7.4, solubilised in LMNG.

SupramoleculeName: Connexin 26, 90mmHg PCO2, pH7.4, solubilised in LMNG. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Gap junction beta-2 protein

MacromoleculeName: Gap junction beta-2 protein / type: protein_or_peptide / ID: 1 / Details: Connexin 26 subunit / 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

UniProtKB: Gap junction beta-2 protein

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Macromolecule #2: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 2 / Number of copies: 13 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 322 / 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

Concentration3.7 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
70.0 mMNaClsodium chloride
5.0 %C3H8O3glycerol
1.0 mMC4H10O2S2DTT
0.003 %C47H88O22LMNG
80.0 mMNaH2P04sodium hydrogen carbonate
3.0 mMKClpotassium chloride
1.0 mMMgSO4magnesium sulphate
4.0 mMMgCl2magnesium chloride

Details: The buffer, except LMNG 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 LMNG 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: FEI VITROBOT MARK I
Details: Blot 3 seconds, force 10, 1 blot, skip transfer. 3ul sample, 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: 11362 / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.3000000000000003 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: 7652471
Startup modelType of model: INSILICO MODEL / In silico model: Started from alphafold model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4)
Details: Particles were symmetry expanded before classification
Number images used: 240137
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 4)
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-8qa2:
Cryo-EM structure of Cx26 solubilised in LMNG: classification on subunit A; Nconst-mon conformation

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