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- EMDB-20914: Human Connexin-26 (Neutral pH open conformation) -

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Basic information

Entry
Database: EMDB / ID: EMD-20914
TitleHuman Connexin-26 (Neutral pH open conformation)
Map dataSharpened map at 4.0 A resolution.
Sample
  • Organelle or cellular component: Connexin-26 Gap Junction Channel
    • Protein or peptide: Gap junction beta-2 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: 4.0 Å
AuthorsKhan AK / Jagielnicki M / Purdy MD / Yeager M
Funding support United States, 2 items
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, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseApr 29, 2020-
UpdateMay 6, 2020-
Current statusMay 6, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uvr
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20914.png

Downloads & links

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Map

FileDownload / File: emd_20914.map.gz / Format: CCP4 / Size: 61 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map at 4.0 A resolution.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 252 pix.
= 267.12 Å		1.06 Å/pix.
x 252 pix.
= 267.12 Å		1.06 Å/pix.
x 252 pix.
= 267.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.04180314 - 0.1145691
Average (Standard dev.)0.00000503189 (±0.0053239064)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions252252252
Spacing252252252
CellA=B=C: 267.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z252252252
origin x/y/z0.0000.0000.000
length x/y/z267.120267.120267.120
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS252252252
D min/max/mean-0.0420.1150.000

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Supplemental data

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Additional map: Not sharpened map at 4.4 A resolution.

Fileemd_20914_additional.map
AnnotationNot sharpened map at 4.4 A resolution.
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 Gap Junction Channel

EntireName: Connexin-26 Gap Junction Channel
Components
  • Organelle or cellular component: Connexin-26 Gap Junction Channel
    • Protein or peptide: Gap junction beta-2 protein

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Supramolecule #1: Connexin-26 Gap Junction Channel

SupramoleculeName: Connexin-26 Gap Junction Channel / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 326 kDa/nm
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Gap junction beta-2 protein

MacromoleculeName: Gap junction beta-2 protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.214945 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP ISHIRLWALQ LIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQKVRIE GSLWWTYTSS IFFRVIFEAA FMYVFYVMYD G FSMQRLVK ...String:
MDWGTLQTIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHYFP ISHIRLWALQ LIFVSTPAL LVAMHVAYRR HEKKRKFIKG EIKSEFKDIE EIKTQKVRIE GSLWWTYTSS IFFRVIFEAA FMYVFYVMYD G FSMQRLVK CNAWPCPNTV DCFVSRPTEK TVFTVFMIAV SGICILLNVT ELSYLLIRYS SGKSKKPV

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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

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
200.0 mMNaClsodium chloride

Details: NaCl was diluted from 1 M to 200 mM immediately before freezing.
GridModel: C-flat-2/2
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: After application of sample to the C-flat holey carbon grid, sample was manually blotted with Whatman no. 1 filter paper for 3-5 s..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 8.0 sec. / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 64087
CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION)
Startup modelType of model: OTHER
Details: 2D class averages of the auto-picked particles were calculated and used to generate a de novo D6 symmetric initial model in EMAN2.
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 10345
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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Atomic model buiding 1

DetailsInitial 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.
RefinementSpace: REAL / Protocol: OTHER / Target criteria: Model-map cross correlation
Output model

PDB-6uvr:
Human Connexin-26 (Neutral pH open conformation)

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