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- EMDB-18296: Cryo-EM reconstruction of Cx26 gap junction K125E mutant in HEPES... -

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Basic information

Entry
Database: EMDB / ID: EMD-18296
TitleCryo-EM reconstruction of Cx26 gap junction K125E mutant in HEPES buffer
Map dataFull map D6 averaged
Sample
  • Complex: Connexin 26 K125E in HEPES buffer pH 8.0
    • Protein or peptide: Connexin 26 K125E
KeywordsGap junction Large Pore Channel Carbon dioxide sensitive / MEMBRANE PROTEIN
Function / homology
Function and homology information


Transport of connexons to the plasma membrane / gap junction-mediated intercellular transport / 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 / gap junction channel activity / Gap junction assembly ...Transport of connexons to the plasma membrane / gap junction-mediated intercellular transport / 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 / gap junction channel activity / Gap junction assembly / endoplasmic reticulum-Golgi intermediate compartment / sensory perception of sound / transmembrane transport / cell-cell signaling / calcium ion binding / 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: 4.3 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
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_18296.png

Downloads & links

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Map

FileDownload / File: emd_18296.map.gz / Format: CCP4 / Size: 122.7 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.)
1.08 Å/pix.
x 318 pix.
= 343.44 Å		1.08 Å/pix.
x 318 pix.
= 343.44 Å		1.08 Å/pix.
x 318 pix.
= 343.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0112
Minimum - Maximum-0.0068910043 - 0.03848601
Average (Standard dev.)-0.0000010124513 (±0.0019026803)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions318318318
Spacing318318318
CellA=B=C: 343.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map D6 averaged

Fileemd_18296_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_18296_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 K125E in HEPES buffer pH 8.0

EntireName: Connexin 26 K125E in HEPES buffer pH 8.0
Components
  • Complex: Connexin 26 K125E in HEPES buffer pH 8.0
    • Protein or peptide: Connexin 26 K125E

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Supramolecule #1: Connexin 26 K125E in HEPES buffer pH 8.0

SupramoleculeName: Connexin 26 K125E in HEPES buffer pH 8.0 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Connexin 26 K125E

MacromoleculeName: Connexin 26 K125E / type: protein_or_peptide / ID: 1 / Details: Connexin 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 EIKTQEVRIE GSLWWTYTSS IFFRV IFEA AFMYVFYVMY DGFSMQRLVK ...String:
MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP IS HIRLWAL QLIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQEVRIE 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 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
250.0 mMNaClsodium chloride
1.0 mMCaCl2calcium chloride
2.5 %C3H8O3glycerol
5.0 mMC4H10O2S2DTT
0.03 %C2H46O11DDM

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.
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: LEICA EM GP
Details: 3 microlitres protein applied to grid, blot time 3 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 2573 / Average exposure time: 44.0 sec. / Average electron dose: 40.7 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: 1.7 µm / Nominal defocus min: 0.3 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: 680463
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 5 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 147546
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3)
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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