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- EMDB-9116: CryoEM reconstruction of native lens connexin-46/50 at 3.4 angstr... -

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Basic information

Entry
Database: EMDB / ID: EMD-9116
TitleCryoEM reconstruction of native lens connexin-46/50 at 3.4 angstrom resolution
Map dataCx46/50 D6 symmetrized map, 3.4 angstrom resolution, sharpened
Sample
  • Complex: Connexin-46 gap junction
    • Protein or peptide: Gap junction alpha-3 protein, connexin-46
Function / homology
Function and homology information


gap junction hemi-channel activity / gap junction-mediated intercellular transport / connexin complex / cell communication / gap junction channel activity / visual perception / 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) / Sheep (sheep)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMyers JB / Reichow SL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM124779 United States
CitationJournal: Nature / Year: 2018
Title: Structure of native lens connexin 46/50 intercellular channels by cryo-EM.
Authors: Janette B Myers / Bassam G Haddad / Susan E O'Neill / Dror S Chorev / Craig C Yoshioka / Carol V Robinson / Daniel M Zuckerman / Steve L Reichow /
Abstract: Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of ...Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which-together with computational studies-elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. 'Hot spots' of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects.
History
DepositionSep 18, 2018-
Header (metadata) releaseOct 10, 2018-
Map releaseDec 12, 2018-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing 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-6mhq
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mhy
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9116.png

Downloads & links

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Map

FileDownload / File: emd_9116.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCx46/50 D6 symmetrized map, 3.4 angstrom resolution, sharpened
Voxel sizeX=Y=Z: 0.665 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.11425026 - 0.18102528
Average (Standard dev.)0.0005660806 (±0.0062425938)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 212.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6650.6650.665
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z212.800212.800212.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-153-266-98
NX/NY/NZ528514389
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1140.1810.001

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Supplemental data

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Mask #1

Fileemd_9116_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_9116_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cx46/50 D6 symmetrized map, 3.5 angstrom resolution, sharpened

Fileemd_9116_additional_1.map
AnnotationCx46/50 D6 symmetrized map, 3.5 angstrom resolution, sharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cx46/50 D6 symmetrized map, 3.5 angstrom resolution, unprocessed

Fileemd_9116_additional_2.map
AnnotationCx46/50 D6 symmetrized map, 3.5 angstrom resolution, unprocessed
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cx46/50 D6 symmetrized map, 3.4 angstrom resolution, unprocessed

Fileemd_9116_additional_3.map
AnnotationCx46/50 D6 symmetrized map, 3.4 angstrom resolution, unprocessed
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-46 gap junction

EntireName: Connexin-46 gap junction
Components
  • Complex: Connexin-46 gap junction
    • Protein or peptide: Gap junction alpha-3 protein, connexin-46

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Supramolecule #1: Connexin-46 gap junction

SupramoleculeName: Connexin-46 gap junction / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Ovis aries (sheep) / Organ: Eye / Tissue: Lens
Location in cell: C-terminal truncated version isolated from lens core
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: Gap junction alpha-3 protein, connexin-46

MacromoleculeName: Gap junction alpha-3 protein, connexin-46 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Sheep (sheep) / Organ: Eye / Tissue: Lens
Molecular weightTheoretical: 37.968516 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 TP

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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

Concentration2.35 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHepes
150.0 mMNaClSodium chloride
2.0 mMEDTAEthylenediaminetetraacetic acid
2.0 mMEGTA
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: 10 sec wait before blotting, 4.0 second blot before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 398066
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: CX46/50 negative stain model generated in EMAN2
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 30128
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2zw3


Chain - Chain ID: A
RefinementSpace: REAL
Output model

PDB-6mhq:
Structure of connexin-46 intercellular gap junction channel at 3.4 angstrom resolution by cryoEM

PDB-6mhy:
Structure of connexin-50 intercellular gap junction channel at 3.4 angstrom resolution by cryoEM

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