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- EMDB-73957: Destabilized open state sheep connexin-46/50 in DMPC nanodiscs at... -

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Basic information

Entry
Database: EMDB / ID: EMD-73957
TitleDestabilized open state sheep connexin-46/50 in DMPC nanodiscs at low pH
Map data
Sample
  • Complex: Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH
    • Protein or peptide: Gap junction alpha-3 protein
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water
Keywordsconnexin / gap junction / cryo-EM / pH regulation / lipid gating / large-pore channel / MEMBRANE PROTEIN
Function / homology
Function and homology information


gap junction-mediated intercellular transport / gap junction hemi-channel activity / connexin complex / gap junction channel activity / visual perception / cell-cell signaling / plasma membrane
Similarity search - Function
Gap junction alpha-3 protein (Cx46) / Gap junction alpha-8 protein (Cx50) / Gap junction alpha-8 protein (Cx50) / Connexin, C-terminal / Connexin / Connexin, N-terminal / Connexin, conserved site / Gap junction protein, cysteine-rich domain / Connexin, N-terminal domain superfamily / Connexin ...Gap junction alpha-3 protein (Cx46) / Gap junction alpha-8 protein (Cx50) / Gap junction alpha-8 protein (Cx50) / Connexin, C-terminal / 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 alpha-8 protein / Gap junction alpha-3 protein
Similarity search - Component
Biological speciesOvis aries (sheep)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsJarodsky JM / Myers JB / Reichow SL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R35GM124779 United States
CitationJournal: Nat Commun / Year: 2026
Title: Reversible lipid-mediated pH-gating of connexin-46/50 by cryo-EM.
Authors: Joshua M Jarodsky / Janette B Myers / Steve L Reichow /
Abstract: Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to ...Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to intracellular pH changes, closing under acidic conditions to limit the spread of cytotoxic signals during cellular stress, such as ischemia. Using cryo-electron microscopy (cryo-EM), we uncover insights into the structural mechanism of pH-gating in native lens connexin-46/50 (Cx46/50) gap junctions. Mild acidification drives lipid infiltration into the channel pore, displacing the N-terminal (NT) domain and stabilizing pore closure. Lipid involvement is shown to be both essential and fully reversible. Structural transitions involve an ensemble of gated states formed through non-cooperative NT domain movement as well as minor populations of a distinct destabilized open-state. These findings provide molecular insights into pH-gating dynamics, illustrating how structural changes may regulate gap junction function under cellular stress and linking Cx46/50 dysregulation to age-related cataract formation.
History
DepositionNov 18, 2025-
Header (metadata) releaseJan 28, 2026-
Map releaseJan 28, 2026-
UpdateJan 28, 2026-
Current statusJan 28, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73957.png

Downloads & links

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Map

FileDownload / File: emd_73957.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.8 Å/pix.
x 384 pix.
= 307.814 Å		0.8 Å/pix.
x 384 pix.
= 307.814 Å		0.8 Å/pix.
x 384 pix.
= 307.814 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8016 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-2.2144663 - 3.3751097
Average (Standard dev.)0.0022200556 (±0.05126485)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 307.8144 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_73957_additional_1.map
Projections & Slices
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Half map: #2

Fileemd_73957_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_73957_half_map_2.map
Projections & Slices
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Sample components

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Entire : Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH

EntireName: Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH
Components
  • Complex: Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH
    • Protein or peptide: Gap junction alpha-3 protein
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water

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Supramolecule #1: Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH

SupramoleculeName: Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Connexin-46 and connexin-50 coassemble in the gap junction complex and arrangement cannot be disentangled. Thus both isoforms are individually modeled into the same gap junction density.
Source (natural)Organism: Ovis aries (sheep)

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Macromolecule #1: Gap junction alpha-3 protein

MacromoleculeName: Gap junction alpha-3 protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Ovis aries (sheep)
Molecular weightTheoretical: 44.033027 KDa
SequenceString: MGDWSFLGRL LENAQEHSTV IGKVWLTVLF IFRILVLGAA AEEVWGDEQS DFTCNTQQPG CENVCYDRAF PISHVRFWVL QIIFVSTPT LIYLGHVLHL VRMEEKRKER EEEPPKAAGP AEEHQDPAPV RDDRGKVRIA GALLRTYVFN IIFKTLFEVG F IAGQYFLY ...String:
MGDWSFLGRL LENAQEHSTV IGKVWLTVLF IFRILVLGAA AEEVWGDEQS DFTCNTQQPG CENVCYDRAF PISHVRFWVL QIIFVSTPT LIYLGHVLHL VRMEEKRKER EEEPPKAAGP AEEHQDPAPV RDDRGKVRIA GALLRTYVFN IIFKTLFEVG F IAGQYFLY GFQLKPLYRC DRWPCPNTVD CFISRPTEKT IFILFMLAVA CVSLLLNVLE IYHLGWKKLK QGMTSPFRPD TP GSRAGSA KPMGGSPLLL PPNSAPPAVT IGFPPYYAPS ASSLGQASAP GYPEPPLPAA LPGTPGTPGT PGTLGGGGGN QGL RAPAQN CANREAEPQT SARKASPPAS TPPAAPAGGP QQFLPGGAAG SSGDSDGEGA VTAVELHAPP EPPADPGRSS KASK SSGGR ARAADLAI

UniProtKB: Gap junction alpha-3 protein

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Macromolecule #2: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 2 / Number of copies: 288 / Formula: MC3
Molecular weightTheoretical: 677.933 Da
Chemical component information

ChemComp-MC3:
1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 576 / 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

BufferpH: 5.8
Component:
ConcentrationFormulaName
20.0 mMC4H6O4Succinate
2.0 mMC10H16N2O8EDTA
2.0 mMC14H24N2O10EGTA
150.0 mMNaClSodium Chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 7072 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 29000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2503619
CTF correctionSoftware - Name: cryoSPARC (ver. v4.4.1) / Type: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.1) / Number images used: 218088
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v4.4.1)

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Atomic model buiding 1

Initial modelPDB ID:

7jkc


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

PDB-9z9s:
Destabilized open state sheep connexin-46 in DMPC nanodiscs at low pH

PDB-9z9w:
Destabilized open state sheep connexin-50 in DMPC nanodiscs at low pH

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