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- PDB-8q9z: Cryo-EM structure of Cx26 gap junction K125E mutant in bicarbonat... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8q9z | |||||||||
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Title | Cryo-EM structure of Cx26 gap junction K125E mutant in bicarbonate buffer (classification on hemichannel) | |||||||||
![]() | Gap junction beta-2 protein | |||||||||
![]() | MEMBRANE PROTEIN / Gap junction Large Pore Channel Carbon dioxide sensitive | |||||||||
Function / homology | ![]() Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / epididymis development / gap junction channel activity involved in cell communication by electrical coupling / 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 / epididymis development / gap junction channel activity involved in cell communication by electrical coupling / 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 / cellular response to glucagon stimulus / inner ear development / decidualization / endoplasmic reticulum-Golgi intermediate compartment / lateral plasma membrane / response to retinoic acid / 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 | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å | |||||||||
![]() | Brotherton, D.H. / Cameron, A.D. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO. Authors: Deborah H Brotherton / Sarbjit Nijjar / Christos G Savva / Nicholas Dale / Alexander David Cameron / ![]() Abstract: Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO. This is proposed to be mediated ...Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation. #1: ![]() Title: Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2 Authors: Brotherton, D.H. / Nijjar, S. / Savva, C.G. / Dale, N. / Cameron, A.D. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 608.8 KB | Display | ![]() |
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PDB format | ![]() | 401.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.7 MB | Display | ![]() |
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Full document | ![]() | 1.7 MB | Display | |
Data in XML | ![]() | 79.9 KB | Display | |
Data in CIF | ![]() | 116 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 18290MC ![]() 8qa0C ![]() 8qa1C ![]() 8qa2C ![]() 8qa3C M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 26713.607 Da / Num. of mol.: 12 / Mutation: K125E Source method: isolated from a genetically manipulated source Details: Connexin 26 subunit / Source: (gene. exp.) ![]() ![]() ![]() #2: Sugar | ChemComp-LMT / #3: Chemical | ChemComp-PTY / #4: Water | ChemComp-HOH / | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Connexin 26 K125E in 90mmHg PCO2, pH7.4 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.4 Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of used. The basal buffer was filtered and degassed and DDM and DTT added. The buffer was pH corrected at point of ...Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of used. The basal buffer was filtered and degassed and DDM and DTT added. The buffer was pH corrected at point of use to pH 7.4 using 15% CO2 in 85% N2. | |||||||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1 | |||||||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE-PROPANE Details: 3 microlitres protein applied to grid, blot time 7 seconds, in 15% CO2/85%N2 atmosphere |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3 sec. / Electron dose: 41 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4731 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1104398 | ||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161625 / Symmetry type: POINT | ||||||||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 7QEQ Accession code: 7QEQ / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 65.46 Å2 | ||||||||||||||||||||||||||||||
Refine LS restraints |
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