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- PDB-6uvs: Human Connexin-26 (Low pH open conformation) -

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Basic information

Entry
Database: PDB / ID: 6uvs
TitleHuman Connexin-26 (Low pH open conformation)
ComponentsGap junction beta-2 protein
KeywordsMEMBRANE PROTEIN / gap junction channel
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 / astrocyte projection / gap junction / 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 progesterone / response to ischemia / sensory perception of sound / transmembrane transport / response to estradiol / cell-cell signaling / cell body / cellular response to oxidative stress / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKhan, A.K. / Jagielnicki, M. / Purdy, M.D. / Yeager, M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 HL48908 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)F30 HL129702-01 United States
CitationJournal: Cell Rep / Year: 2020
Title: A Steric "Ball-and-Chain" Mechanism for pH-Mediated Regulation of Gap Junction Channels.
Authors: Ali K Khan / Maciej Jagielnicki / William E McIntire / Michael D Purdy / Venkatasubramanian Dharmarajan / Patrick R Griffin / Mark Yeager /
Abstract: Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH ...Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric "ball-and-chain" gating particle.
History
DepositionNov 4, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Gap junction beta-2 protein
K: Gap junction beta-2 protein
L: Gap junction beta-2 protein
B: Gap junction beta-2 protein
H: Gap junction beta-2 protein
C: Gap junction beta-2 protein
I: Gap junction beta-2 protein
D: Gap junction beta-2 protein
J: Gap junction beta-2 protein
E: Gap junction beta-2 protein
F: Gap junction beta-2 protein
G: Gap junction beta-2 protein


Theoretical massNumber of molelcules
Total (without water)314,57912
Polymers314,57912
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Gap junction beta-2 protein / Connexin-26 / Cx26


Mass: 26214.945 Da / Num. of mol.: 12 / Mutation: C211S, C218S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GJB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P29033

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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: Connexin-26 Gap Junction Channel / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 326 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 6.4
Details: NaCl was diluted from 1 M to 200 mM immediately before freezing. HEPES pH 7.5 buffer was diluted with MES pH 6.0 buffer thus adjusting pH to 6.4.
Buffer component
IDConc.NameFormulaBuffer-ID
112.5 mMHEPESC8H18N2O4S1
237.5 mMMESC6H13NO4S1
3200 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: After application of sample to the C-flat holey carbon grid, sample was blotted with Whatman no. 1 filter paper for ~4s, blot force of 3, and 100 % humidity at 22oC.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Cs: 2.7 mm
Image recordingAverage exposure time: 10 sec. / Electron dose: 45 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 25

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
5RELIONCTF correction
8UCSF Chimeramodel fittingInitial rigid docking
10PHENIXmodel refinementReal space refine
11Cootmodel refinementManual refinement
12RELIONinitial Euler assignment
13RELIONfinal Euler assignment
15RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 49952
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25236 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Model-map cross correlation
Details: Initial local fitting was done using UCSF Chimera. Multiple rounds of automated (Phenix) and manual (Coot) model building were performed. The model was validated in Molprobity(part of Phenix ...Details: Initial local fitting was done using UCSF Chimera. Multiple rounds of automated (Phenix) and manual (Coot) model building were performed. The model was validated in Molprobity(part of Phenix package). Model-map cross correlation score as well as EMRinger score were obtained in Phenix. RMSD values for C-alphas were calculated in UCSF Chimera.

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