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- EMDB-22390: Sheep Connexin-50 at 2.5 angstroms reoslution, Lipid Class 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-22390
TitleSheep Connexin-50 at 2.5 angstroms reoslution, Lipid Class 2
Map dataLocal resolution-filtered map. Used for model-building.
Sample
  • Complex: Dodecameric Connexin-50 Gap Junction Channel Complex
    • Protein or peptide: Gap junction alpha-8 protein
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water
KeywordsConnexin / Gap Junction / Lipid / Nanodisc / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsFlores JA / Haddad BG
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 JA / Yoshioka CC / Copperman J / Zuckerman DM / Reichow SL
History
DepositionJul 31, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing 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
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  • 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-7jm9
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7jn0
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22390.png

Downloads & links

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Map

FileDownload / File: emd_22390.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution-filtered map. Used for model-building.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 400 pix.
= 259.6 Å		0.65 Å/pix.
x 400 pix.
= 259.6 Å		0.65 Å/pix.
x 400 pix.
= 259.6 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.649 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.25673327 - 0.39569733
Average (Standard dev.)0.00006725922 (±0.011949778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 259.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6490.6490.649
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z259.600259.600259.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.2570.3960.000

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Supplemental data

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Mask #1

Fileemd_22390_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Postprocessed map, unmasked.

Fileemd_22390_additional_1.map
AnnotationPostprocessed map, unmasked.
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Histogram

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Additional map: Postprocessed map, masked.

Fileemd_22390_additional_2.map
AnnotationPostprocessed map, masked.
Projections & Slices
AxesZYX

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Additional map: Postprocessed map, lowpass-filtered to 3.5 angstroms. Used to...

Fileemd_22390_additional_3.map
AnnotationPostprocessed map, lowpass-filtered to 3.5 angstroms. Used to aid lipid modeling.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Pre-postprocessed map.

Fileemd_22390_additional_4.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 pre-postprocessed, postprocessed,...

Fileemd_22390_half_map_1.map
AnnotationHalf-map (even) used for calculation of pre-postprocessed, postprocessed, and local resolution-filtered maps.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map (odd) used for calculation of pre-postprocessed, postprocessed,...

Fileemd_22390_half_map_2.map
AnnotationHalf-map (odd) used for calculation of pre-postprocessed, postprocessed, and local resolution-filtered maps.
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 Complex

EntireName: Dodecameric Connexin-50 Gap Junction Channel Complex
Components
  • Complex: Dodecameric Connexin-50 Gap Junction Channel Complex
    • 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 Complex

SupramoleculeName: Dodecameric Connexin-50 Gap Junction Channel Complex / 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: Ovis aries (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

UniProtKB: Gap junction alpha-8 protein

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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: 168 / 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: 156 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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
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: 30.0 sec. / Average electron dose: 52.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 9188
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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