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- EMDB-18295: Cryo-EM reconstruction of Cx26 gap junction K125R mutant (D6 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-18295
TitleCryo-EM reconstruction of Cx26 gap junction K125R mutant (D6 symmetry)
Map dataFull map D6 averaged
Sample
  • Complex: Connexin 26 K125R, 90mmHg PCO2, pH7.4, solubilised in DDM.
    • Protein or peptide: Connexin 26 dodecamer
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 / 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: 2.1 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
CitationJournal: 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.
History
DepositionAug 22, 2023-
Header (metadata) releaseJun 12, 2024-
Map releaseJun 12, 2024-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18295.png

Downloads & links

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Map

FileDownload / File: emd_18295.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.)
0.84 Å/pix.
x 436 pix.
= 364.06 Å		0.84 Å/pix.
x 436 pix.
= 364.06 Å		0.84 Å/pix.
x 436 pix.
= 364.06 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0032
Minimum - Maximum-0.0082749 - 0.024206562
Average (Standard dev.)0.000014631752 (±0.00055631134)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: Half map D6 averaged

Fileemd_18295_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_18295_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 K125R, 90mmHg PCO2, pH7.4, solubilised in DDM.

EntireName: Connexin 26 K125R, 90mmHg PCO2, pH7.4, solubilised in DDM.
Components
  • Complex: Connexin 26 K125R, 90mmHg PCO2, pH7.4, solubilised in DDM.
    • Protein or peptide: Connexin 26 dodecamer

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Supramolecule #1: Connexin 26 K125R, 90mmHg PCO2, pH7.4, solubilised in DDM.

SupramoleculeName: Connexin 26 K125R, 90mmHg PCO2, pH7.4, solubilised in DDM.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Connexin 26 dodecamer

MacromoleculeName: Connexin 26 dodecamer / type: protein_or_peptide / ID: 1 / Details: Connexin 26 subunit / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP IS HIRLWAL QLIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQRVRIE GSLWWTYTSS IFFRV IFEA AFMYVFYVMY DGFSMQRLVK ...String:
MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP IS HIRLWAL QLIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQRVRIE GSLWWTYTSS IFFRV IFEA AFMYVFYVMY DGFSMQRLVK CNAWPCPNTV DCFVSRPTEK TVFTVFMIAV SGICILLNVT ELCYLLIR Y CSGKSKKPVL VPR

UniProtKB: Gap junction beta-2 protein

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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
0.03 %C2H46O11DDM
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.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
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: 10044 / Average electron dose: 36.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 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: 2382993
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 5 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 222622
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 6 / 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

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