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- PDB-9qnd: Connexin-32 (Cx32) W3S mutant gap junction channel in POPC-contai... -

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Basic information

Entry
Database: PDB / ID: 9qnd
TitleConnexin-32 (Cx32) W3S mutant gap junction channel in POPC-containing MSP2N2 nanodiscs
ComponentsGap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
KeywordsMEMBRANE PROTEIN / Ion channel / gap junction / membrane transport
Function / homology
Function and homology information


Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / Gap junction assembly / gap junction channel activity / nervous system development / cell-cell signaling / endoplasmic reticulum membrane / identical protein binding
Similarity search - Function
Gap junction beta-1 protein (Cx32) / 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-1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.35 Å
AuthorsKorkhov, V.M. / Lavriha, P. / Fluri, C.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation184951 Switzerland
CitationJournal: Nat Commun / Year: 2025
Title: Lipid dependence of connexin-32 gap junction channel conformations.
Authors: Pia Lavriha / Carina Fluri / Jorge Enrique Hernández González / Volodymyr M Korkhov /
Abstract: Connexin-32 (Cx32) gap junction channels (GJCs) mediate intercellular coupling in various tissues, including myelinating Schwann cells. Mutations in Cx32, such as W3S, are associated with X-linked ...Connexin-32 (Cx32) gap junction channels (GJCs) mediate intercellular coupling in various tissues, including myelinating Schwann cells. Mutations in Cx32, such as W3S, are associated with X-linked Charcot-Marie-Tooth (CMT1X) disease. Lipids regulate Cx32 GJC permeation, although the regulatory mechanism is unclear. Here, we determine the cryo-EM structures of Cx32 GJCs reconstituted in nanodiscs, revealing that phospholipids block the Cx32 GJC pore by binding to the site formed by N-terminal gating helices. The phospholipid-bound state is contingent on the presence of a sterol molecule in a hydrophobic pocket formed by the N-terminus: the N-terminal helix of Cx32 fails to sustain a phospholipid binding site in the absence of cholesterol hemisuccinate. The CMT1X-linked W3S mutant which has an impaired sterol binding site adopts a conformation of the N-terminus incompatible with phospholipid binding. Our results indicate that different lipid species control connexin channel gating directly by influencing the conformation of the N-terminal gating helix.
History
DepositionMar 24, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
B: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
C: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
D: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
E: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
F: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
G: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
H: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
I: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
J: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
K: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein
L: Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)758,74012
Polymers758,74012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Gap junction beta-1 protein,Gap junction beta-1 protein,Green fluorescent protein / Connexin-32 / Cx32 / GAP junction 28 kDa liver protein


Mass: 63228.297 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Aequorea victoria (jellyfish)
Gene: GJB1, CX32, GFP / Plasmid: pACMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P08034
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: A dodecameric complex of connexin-32 (Cx32) W3S mutant gap junction channel
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 / Cell: HEK293 / Plasmid: pACMV
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION4.0.1particle selection
2PHENIX1.21.2_5419:model refinement
5CTFFIND4CTF correction
10RELION4.0.1initial Euler assignment
11RELION4.0.1final Euler assignment
12RELION4.0.1classification
13RELION4.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 2.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68649 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318960
ELECTRON MICROSCOPYf_angle_d0.46825872
ELECTRON MICROSCOPYf_dihedral_angle_d10.4826528
ELECTRON MICROSCOPYf_chiral_restr0.043108
ELECTRON MICROSCOPYf_plane_restr0.0033084

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