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- PDB-3iz1: C-alpha model fitted into the EM structure of Cx26M34A -

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Entry
Database: PDB / ID: 3iz1
TitleC-alpha model fitted into the EM structure of Cx26M34A
ComponentsGap junction beta-2 protein
KeywordsMEMBRANE PROTEIN / Gap Junction channel
Function / homology
Function and homology information


Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / epididymis development / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction ...Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / epididymis development / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction / astrocyte projection / Gap junction assembly / gap junction channel activity / endoplasmic reticulum-Golgi intermediate compartment / inner ear development / decidualization / lateral plasma membrane / response to retinoic acid / cellular response to glucagon stimulus / cellular response to dexamethasone stimulus / response to ischemia / response to progesterone / sensory perception of sound / transmembrane transport / cell-cell signaling / response to estradiol / cellular response to oxidative stress / cell body / response to lipopolysaccharide / calcium ion binding / perinuclear region of cytoplasm / identical protein binding / plasma membrane
Similarity search - Function
Gap junction beta-2 protein (Cx26) / 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 beta-2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 6 Å
AuthorsOshima, A. / Tani, K. / Toloue, M.M. / Hiroaki, Y. / Smock, A. / Inukai, S. / Cone, A. / Nicholson, B.J. / Sosinsky, G.E. / Fujiyoshi, Y.
CitationJournal: J Mol Biol / Year: 2011
Title: Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.
Authors: Atsunori Oshima / Kazutoshi Tani / Masoud M Toloue / Yoko Hiroaki / Amy Smock / Sayaka Inukai / Angela Cone / Bruce J Nicholson / Gina E Sosinsky / Yoshinori Fujiyoshi /
Abstract: Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we ...Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration.
History
DepositionAug 19, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 3, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jul 18, 2018Group: Author supporting evidence / Data collection
Category: em_image_scans / em_single_particle_entity / em_software
Item: _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._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 / _struct_ref_seq_dif.details

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Assembly

Deposited unit
A: Gap junction beta-2 protein
B: Gap junction beta-2 protein
C: Gap junction beta-2 protein


Theoretical massNumber of molelcules
Total (without water)83,0603
Polymers83,0603
Non-polymers00
Water00
1
A: Gap junction beta-2 protein
B: Gap junction beta-2 protein
C: Gap junction beta-2 protein

A: Gap junction beta-2 protein
B: Gap junction beta-2 protein
C: Gap junction beta-2 protein


Theoretical massNumber of molelcules
Total (without water)166,1206
Polymers166,1206
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
Unit cell
Length a, b, c (Å)112.400, 111.200, 300.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Gap junction beta-2 protein / Connexin-26 / Cx26 / Coordinate model: Cα atoms only


Mass: 27686.686 Da / Num. of mol.: 3 / Mutation: M34A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GJB2 / Plasmid: pBlueBac4.5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P29033

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: human connexin26 M34A mutant / Type: COMPLEX
Buffer solutionName: 10mM MES / pH: 5.8 / Details: 10mM MES
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: REICHERT-JUNG PLUNGER / Cryogen name: NITROGEN / Temp: 93 K
Method: The grids were blotted with filter paper and fast frozen into liquid nitrogen

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Data collection

MicroscopyModel: JEOL KYOTO-3000SFF / Date: Oct 3, 2008
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 40000 X / Calibrated magnification: 39000 X / Nominal defocus max: 3939 nm / Nominal defocus min: 458 nm / Cs: 1.6 mm
Specimen holderTemperature: 4.2 K / Tilt angle max: 60 ° / Tilt angle min: 0 °
Image recordingElectron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Situsmodel fitting
2MRC3D reconstruction
CTF correctionDetails: Each image
3D reconstructionMethod: Lattice line fitting / Resolution: 6 Å / Magnification calibration: carbon grating / Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--Local refinement, Flexible fitting REFINEMENT PROTOCOL--rigid body DETAILS--Initial local fitting was done using O, and Situs was used for flexible fitting.
Atomic model buildingPDB-ID: 2ZW3

2zw3


Accession code: 2ZW3 / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms555 0 0 0 555

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