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- PDB-7jmc: Sheep Connexin-50 at 2.5 angstroms resolution, Lipid Class 3 -

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Basic information

Entry
Database: PDB / ID: 7jmc
TitleSheep Connexin-50 at 2.5 angstroms resolution, Lipid Class 3
ComponentsGap junction alpha-8 protein
KeywordsMEMBRANE PROTEIN / Connexin / Gap Junction / Lipid / Nanodisc
Function / homology
Function and homology information


gap junction-mediated intercellular transport / connexin complex / cell communication / gap junction channel activity / plasma membrane
Similarity search - Function
Gap junction alpha-8 protein (Cx50) / Gap junction alpha-8 protein (Cx50) / 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. ...Gap junction alpha-8 protein (Cx50) / Gap junction alpha-8 protein (Cx50) / 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
1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / Gap junction alpha-8 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.D. / 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: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
#2: 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 23, 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 / em_admin / 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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Gap junction alpha-8 protein
B: Gap junction alpha-8 protein
C: Gap junction alpha-8 protein
D: Gap junction alpha-8 protein
E: Gap junction alpha-8 protein
F: Gap junction alpha-8 protein
G: Gap junction alpha-8 protein
H: Gap junction alpha-8 protein
I: Gap junction alpha-8 protein
J: Gap junction alpha-8 protein
K: Gap junction alpha-8 protein
L: Gap junction alpha-8 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)688,224156
Polymers590,60212
Non-polymers97,622144
Water3,027168
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(0.500010569519, 0.866019301383, -7.44306248179E-7), (0.866019301384, -0.500010569519, 2.35582255969E-7), (-1.68142210316E-7, -7.62377195039E-7, -1)-17.8995274778, 31.0111166704, 160.952072786
2given(-0.500013602592, 0.866017550182, 2.10907174875E-7), (0.866017550182, 0.500013602592, -3.22450657234E-7), (-3.84704384554E-7, 2.14196001192E-8, -1)29.4756241433, -17.0128308106, 160.952040023
3given(-0.999999999999, 1.49284998076E-7, -1.43874028682E-6), (1.49284340836E-7, 1, 4.56816955284E-7), (1.43874035502E-6, 4.56816740502E-7, -0.999999999999)94.754227222, -8.69395711121E-5, 160.951963381
4given(-0.500013315275, -0.866017716069, -1.28775103976E-6), (-0.866017716069, 0.500013315276, -5.63765391569E-7), (1.13212348345E-6, 8.33325011846E-7, -0.999999999999)112.662139163, 65.0437770168, 160.952003416
5given(0.50000969889, -0.866019804055, -5.38226302083E-7), (-0.866019804055, -0.500009698889, -6.13789890787E-7), (2.62435829711E-7, 7.73015535141E-7, -1)65.2801626224, 113.074562022, 160.952011349
6given(-0.999999999999, 1.03919146472E-6, -6.79887902088E-7), (-1.03919129945E-6, -0.999999999999, -2.43073150038E-7), (-6.79888154687E-7, -2.43072443504E-7, 1)94.7540299929, 96.0520390462, 7.81891486668E-6
7given(-0.500013406846, -0.866017663199, -1.36515043583E-6), (0.8660176632, -0.500013406846, -1.93403083553E-7), (-5.15103033803E-7, -1.27894852505E-6, 0.999999999999)112.659380834, 31.014326225, 4.91226420252E-6
8given(0.500007478459, -0.866021086051, -9.8146769848E-8), (0.866021086051, 0.500007478459, 3.01056735289E-7), (-2.11647361948E-7, -2.35527791301E-7, 1)65.2842798915, -17.0164931197, -4.235305098926E-5
9given(0.999999999999, 1.31422273604E-6, 8.33801658278E-7), (1.31422285818E-6, -0.999999999999, -1.46489282765E-7), (8.33801465758E-7, 1.46490378566E-8, -1)-0.000174499824116, 96.0520042044, 160.95197962
10given(0.500012992509, 0.866017902425, 7.31004073995E-7), (-0.866017902426, 0.500012992509, 6.24102738888E-8), (1.74972610255E-7, -9.4512209293E-7, 1)-17.9022954073, 65.0460374194, -5.30176902203E-6
11given(-0.500012334797, 0.866018282169, 7.92161671165E-8), (-0.866018282169, -0.500012334797, 5.82279674068E-7), (5.43873903752E-7, 2.22544370369E-7, 1)29.4797567531, 113.069997024, -5.75778779677E-5

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Components

#1: Protein
Gap junction alpha-8 protein / Connexin-49 / Cx49 / Lens fiber protein MP70 / MP38 / MP64


Mass: 49216.809 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: P55917
#2: Chemical...
ChemComp-MC3 / 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE


Mass: 677.933 Da / Num. of mol.: 144 / 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.: 168 / 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: Dodecameric Connexin-50 Gap Junction Channel Complex / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.45 MDa / Experimental value: YES
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: 150 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_3928refinement
PHENIXdev_3928refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
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: 6942 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MHY

6mhy


Accession code: 6MHY / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.89 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008721480
ELECTRON MICROSCOPYf_angle_d0.742728440
ELECTRON MICROSCOPYf_chiral_restr0.05633012
ELECTRON MICROSCOPYf_plane_restr0.0063288
ELECTRON MICROSCOPYf_dihedral_angle_d15.36388352

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