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- PDB-8qa0: Cryo-EM structure of Cx26 solubilised in LMNG - hemichannel class... -

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Basic information

Entry
Database: PDB / ID: 8qa0
TitleCryo-EM structure of Cx26 solubilised in LMNG - hemichannel classification - NConst conformation
ComponentsGap junction beta-2 protein
KeywordsMEMBRANE PROTEIN / Gap junction Large Pore Channel Carbon dioxide sensitive
Function / homology
Function and homology information


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
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
PHOSPHATIDYLETHANOLAMINE / Gap junction beta-2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsBrotherton, D.H. / Cameron, A.D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P010393/1 United Kingdom
Leverhulme TrustRPG-2015-090 United Kingdom
Citation
Journal: Elife / Year: 2024
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: Journal: Elife / Year: 2024
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
DepositionAug 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 12, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
D: Gap junction beta-2 protein
E: Gap junction beta-2 protein
F: Gap junction beta-2 protein
G: Gap junction beta-2 protein
H: Gap junction beta-2 protein
I: Gap junction beta-2 protein
J: Gap junction beta-2 protein
K: Gap junction beta-2 protein
L: Gap junction beta-2 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)338,18136
Polymers320,56412
Non-polymers17,61724
Water6,161342
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Gap junction beta-2 protein / Connexin-26 / Cx26


Mass: 26713.674 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Details: Connexin 26 subunit / Source: (gene. exp.) Homo sapiens (human) / Gene: GJB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P29033
#2: Chemical...
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 342 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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 26, 90mmHg PCO2, pH7.4, solubilised in LMNG. / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Details: The buffer, except LMNG 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 LMNG 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
IDConc.NameFormulaBuffer-ID
170 mMsodium chlorideNaCl1
25 %glycerolC3H8O31
31 mMDTTC4H10O2S21
40.003 %LMNGC47H88O221
580 mMsodium hydrogen carbonateNaH2P041
63 mMpotassium chlorideKCl1
71 mMmagnesium sulphateMgSO41
84 mMmagnesium chlorideMgCl21
SpecimenConc.: 3.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE-PROPANE
Details: Blot 3 seconds, force 10, 1 blot, skip transfer. 3ul sample, in 15% CO2/85%N2 atmosphere

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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 / Nominal magnification: 105000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 800 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 11362
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
4CTFFIND4CTF correction
7Coot0.9.8.4model fitting
9PHENIX1.20.1model refinement
12RELION4classification
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7652471
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35007 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7QEQ

7qeq


Accession code: 7QEQ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 77.07 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003520052
ELECTRON MICROSCOPYf_angle_d0.567227132
ELECTRON MICROSCOPYf_chiral_restr0.04023054
ELECTRON MICROSCOPYf_plane_restr0.00313210
ELECTRON MICROSCOPYf_dihedral_angle_d7.8772874

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