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- EMDB-22358: Connexin-46/50 in a dynamic lipid environment resolved by CryoEM ... -

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Basic information

Entry
Database: EMDB / ID: EMD-22358
TitleConnexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 angstroms
Map dataHalf-map (odd) used for calculation of local resolution-filtered and postprocessed maps.
Sample
  • Complex: Dodecameric Connexin-50 gap junction channel in MSP1E1-lipid Nanodisc
    • Protein or peptide: Gap junction alpha-8 protein
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water
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: 1.94 Å
AuthorsFlores JA / Haddad BG / Dolan KD / Myers JB / Yoshioka CC / Copperman J / Zuckerman DM / Reichow SL
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R35GM124779 United States
National Institutes of Health/National Eye Institute (NIH/NEI)1F31EY030409-01 United States
Citation
Journal: Nat Commun / Year: 2020
Title: Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 Å.
Authors: Jonathan A Flores / Bassam G Haddad / Kimberly A Dolan / Janette B Myers / Craig C Yoshioka / Jeremy Copperman / Daniel M Zuckerman / Steve L Reichow /
Abstract: Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel ...Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels.
#1: Journal: BioRxiv / Year: 2020
Title: Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 angstroms
Authors: Flores JA / Haddad BG / Dolan KA / Myers JB / Yoshioka CC / Copperman J / Zuckerman DM / Reichow SL
History
DepositionJul 27, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateSep 9, 2020-
Current statusSep 9, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jjp
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7jkc
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22358.png

Downloads & links

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Map

FileDownload / File: emd_22358.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHalf-map (odd) used for calculation of local resolution-filtered and postprocessed maps.
Voxel sizeX=Y=Z: 0.649 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.1989036 - 0.44592223
Average (Standard dev.)0.0008253871 (±0.016456824)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 194.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6490.6490.649
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z194.700194.700194.700
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1990.4460.001

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Supplemental data

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Mask #1

Fileemd_22358_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocessed map, unmasked.

Fileemd_22358_additional_1.map
AnnotationPostprocessed map, unmasked.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocessed map, masked.

Fileemd_22358_additional_2.map
AnnotationPostprocessed map, masked.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Pre-postprocessed map.

Fileemd_22358_additional_3.map
AnnotationPre-postprocessed map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map (even) used for calculation of local resolution-filtered...

Fileemd_22358_half_map_1.map
AnnotationHalf-map (even) used for calculation of local resolution-filtered and postprocessed maps.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Postprocessed map, unmasked.

Fileemd_22358_half_map_2.map
AnnotationPostprocessed map, unmasked.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dodecameric Connexin-50 gap junction channel in MSP1E1-lipid Nanodisc

EntireName: Dodecameric Connexin-50 gap junction channel in MSP1E1-lipid Nanodisc
Components
  • Complex: Dodecameric Connexin-50 gap junction channel in MSP1E1-lipid Nanodisc
    • Protein or peptide: Gap junction alpha-8 protein
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water

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Supramolecule #1: Dodecameric Connexin-50 gap junction channel in MSP1E1-lipid Nanodisc

SupramoleculeName: Dodecameric Connexin-50 gap junction channel in MSP1E1-lipid Nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Ovis aries (sheep) / Organ: Eye / Tissue: Lens
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: Gap junction alpha-8 protein

MacromoleculeName: Gap junction alpha-8 protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Sheep (sheep) / Organ: Eye / Tissue: Lens
Molecular weightTheoretical: 49.216809 KDa
SequenceString: MGDWSFLGNI LEEVNEHSTV IGRVWLTVLF IFRILILGTA AEFVWGDEQS DFVCNTQQPG CENVCYDEAF PISHIRLWVL QIIFVSTPS LVYVGHAVHH VRMEEKRKER EAEELSQQSP GNGGERAPLA ADQGSVKKSS SSSKGTKKFR LEGTLLRTYV C HIIFKTLF ...String:
MGDWSFLGNI LEEVNEHSTV IGRVWLTVLF IFRILILGTA AEFVWGDEQS DFVCNTQQPG CENVCYDEAF PISHIRLWVL QIIFVSTPS LVYVGHAVHH VRMEEKRKER EAEELSQQSP GNGGERAPLA ADQGSVKKSS SSSKGTKKFR LEGTLLRTYV C HIIFKTLF EVGFIVGHYF LYGFRILPLY RCSRWPCPNV VDCFVSRPTE KTIFILFMLS VASVSLFLNI LEMSHLGLKK IR SAFKRPV EQPLGEIPEK SLHSIAVSSI QKAKGYQLLE EEKIVSHYFP LTEVGMVEAS PLSAKPFSQF EEKVGPGPLG DLS RAYQET LPSYAQVGAQ EGVEEEQPIE AAAEPEVGDK SQEAERVSTE GEETLAVLEE EKVEPPEVEK EAEKEETPPE KVSK QELTP EKAPSLCAEL PGEDTRPLSR LSKASSRARS DDLTV

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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: 180 / 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: 396 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2088 / Average exposure time: 150.0 sec. / Average electron dose: 52.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 26005
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mhy

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7jjp:
Sheep Connexin-50 at 1.9 angstroms resolution by CryoEM

PDB-7jkc:
Sheep Connexin-46 at 1.9 angstroms resolution by CryoEM

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