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- PDB-7jmd: Sheep Connexin-46 at 2.5 angstroms resolution, Lipid Class 1 -

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Basic information

Entry
Database: PDB / ID: 7jmd
TitleSheep Connexin-46 at 2.5 angstroms resolution, Lipid Class 1
ComponentsGap junction alpha-3 protein
KeywordsMEMBRANE PROTEIN / Connexin / Gap Junction / Lipid / Nanodisc
Function / homology
Function and homology information


gap junction-mediated intercellular transport / gap junction hemi-channel activity / connexin complex / visual perception / cell-cell signaling / plasma membrane
Similarity search - Function
Gap junction alpha-3 protein (Cx46) / Gap junction channel protein cysteine-rich domain / 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. ...Gap junction alpha-3 protein (Cx46) / Gap junction channel protein cysteine-rich domain / 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 / de novo design (two linked rop proteins) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / Gap junction alpha-3 protein
Similarity search - Component
Biological speciesOvis aries (sheep)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsFlores, J.A. / Haddad, B.G. / Dolan, K.A. / Myers, J.B. / Yoshioka, C.C. / Copperman, J. / Zuckerman, D.M. / Reichow, S.L.
Funding support United States, 2items
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, J.A. / Haddad, B.G. / Dolan, K.A. / Myers, J.B. / Yoshioka, C.C. / Copperman, J. / Zuckerman, D.M. / Reichow, S.L.
History
DepositionJul 31, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 9, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Gap junction alpha-3 protein
B: Gap junction alpha-3 protein
C: Gap junction alpha-3 protein
D: Gap junction alpha-3 protein
E: Gap junction alpha-3 protein
F: Gap junction alpha-3 protein
G: Gap junction alpha-3 protein
H: Gap junction alpha-3 protein
I: Gap junction alpha-3 protein
J: Gap junction alpha-3 protein
K: Gap junction alpha-3 protein
L: Gap junction alpha-3 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)617,883144
Polymers528,39612
Non-polymers89,487132
Water3,675204
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1, 1.80473623353E-7, -4.03289444269E-8), (1.80473672435E-7, -0.999999999999, 1.21705106944E-6), (-4.03287247813E-8, -1.21705107672E-6, -0.999999999999)0.00302396298289, 102.54748594, 163.548048965
2given(0.499977731133, 0.866038260338, -1.15453734996E-6), (0.866038260339, -0.499977731134, -4.42572888546E-7), (-9.60528019212E-7, -7.78596929379E-7, -0.999999999999)-19.4183510272, 33.6326372378, 163.548057978
3given(-0.500011267067, 0.866018898642, -9.59586451747E-7), (0.866018898641, 0.500011267068, 1.30203459144E-6), (1.60739060047E-6, -1.79988036261E-7, -0.999999999999)30.5554064456, -17.6455491315, 163.547961237
4given(-1, -3.85562410013E-7, 3.67802770719E-7), (-3.85562866658E-7, 0.999999999999, -1.24154922013E-6), (-3.67802292024E-7, -1.24154936194E-6, -0.999999999999)99.95135975, -0.00249122831386, 163.548059265
5given(-0.499978094977, -0.866038050286, 6.23355011541E-7), (-0.866038050286, 0.499978094977, -3.69250601755E-7), (8.12122004538E-9, -7.24466371265E-7, -1)119.366008633, 68.9192599587, 163.548028231
6given(0.500011267067, -0.866018898642, 9.59586299317E-7), (0.866018898641, 0.500011267068, 1.30203470302E-6), (-1.60739062088E-6, 1.79987848463E-7, 0.999999999999)69.3905935545, -17.6455491315, 3.8762717665E-5
7given(0.500011267067, -0.866018898642, 9.59586533369E-7), (-0.866018898641, -0.500011267068, -1.30203438177E-6), (1.60739045971E-6, -1.7998821178E-7, -0.999999999999)69.3905935544, 120.187549131, 163.547961237
8given(0.499976996632, 0.866038684377, -6.57004433961E-7), (-0.866038684378, 0.499976996632, 3.32005876071E-8), (3.57240096878E-7, 5.5239172554E-7, 1)-19.4146257368, 68.9151513715, -3.18888078823E-5
9given(-0.500011267067, 0.866018898642, -9.59586351156E-7), (-0.866018898641, -0.500011267068, -1.3020345179E-6), (-1.60739048649E-6, 1.79987985916E-7, 0.999999999999)30.5554064456, 120.187549131, 3.87627085985E-5
10given(-1, -3.80042284576E-7, 1.11281960855E-7), (3.80042436038E-7, -0.999999999999, 1.36107258356E-6), (1.11281443589E-7, 1.36107262585E-6, 0.999999999999)99.948989574, 102.550260022, -5.66467234933E-5
11given(-0.499977451557, -0.866038421742, 8.56477512639E-7), (0.866038421743, -0.499977451557, 2.45700621811E-7), (2.15433265351E-7, 8.64587204043E-7, 1)119.370446487, 33.6284738216, -4.01107463688E-5

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Components

#1: Protein
Gap junction alpha-3 protein / Connexin-44 / Cx44


Mass: 44033.027 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / Organ: Eye / Plasmid details: Young Sheep / Tissue: Lens / References: UniProt: Q9TU17
#2: Chemical...
ChemComp-MC3 / 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE


Mass: 677.933 Da / Num. of mol.: 132 / Source method: obtained synthetically / Formula: C36H72NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 204 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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-46 / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)Organism: Ovis aries (sheep) / Organ: Eye / Tissue: Lens
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 30 sec. / Electron dose: 52.5 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2088
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_3742refinement
PHENIXdev_3742refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6073 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6MHQ

6mhq


Accession code: 6MHQ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 41.97 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009221828
ELECTRON MICROSCOPYf_angle_d0.931228908
ELECTRON MICROSCOPYf_chiral_restr0.05513000
ELECTRON MICROSCOPYf_plane_restr0.00523336
ELECTRON MICROSCOPYf_dihedral_angle_d19.66578568

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