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- PDB-7qew: human Connexin 26 class 2 hexamer at 90mmHg PCO2, pH7.4 -

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Basic information

Entry
Database: PDB / ID: 7qew
Titlehuman Connexin 26 class 2 hexamer at 90mmHg PCO2, pH7.4
ComponentsGap junction beta-2 protein
KeywordsMEMBRANE PROTEIN / Gap junction / Ion 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: 2.1 Å
AuthorsBrotherton, D.H. / Cameron, A.D. / Savva, C.G. / Ragan, T.J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P010393/1 United Kingdom
Leverhulme TrustRPG-2015-090 United Kingdom
CitationJournal: Structure / Year: 2022
Title: Conformational changes and CO-induced channel gating in connexin26.
Authors: Deborah H Brotherton / Christos G Savva / Timothy J Ragan / Nicholas Dale / Alexander D Cameron /
Abstract: Connexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. ...Connexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. Opening or closing of the channels is controlled by a variety of stimuli, and dysregulation leads to multiple diseases. An increase in the partial pressure of carbon dioxide (PCO) has been shown to cause connexin26 (Cx26) gap junctions to close. Here, we use cryoelectron microscopy (cryo-EM) to determine the structure of human Cx26 gap junctions under increasing levels of PCO. We show a correlation between the level of PCO and the size of the aperture of the pore, governed by the N-terminal helices that line the pore. This indicates that CO alone is sufficient to cause conformational changes in the protein. Analysis of the conformational states shows that movements at the N terminus are linked to both subunit rotation and flexing of the transmembrane helices.
History
DepositionDec 3, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2May 18, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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)174,14136
Polymers157,4826
Non-polymers16,65930
Water4,216234
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27930 Å2
ΔGint-8 kcal/mol
Surface area54500 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "I"
d_2ens_1chain "H"
d_3ens_1chain "J"
d_4ens_1chain "G"
d_5ens_1chain "K"
d_6ens_1chain "L"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ASPTYRM1 - 188
d_12ens_1LMTLMTN
d_13ens_1LMTLMTO
d_14ens_1LMTLMTP
d_15ens_1LMTLMTQ
d_16ens_1PTYPTYR
d_21ens_1ASPTYRG1 - 188
d_22ens_1LMTLMTH
d_23ens_1LMTLMTI
d_24ens_1LMTLMTJ
d_25ens_1LMTLMTK
d_26ens_1PTYPTYL
d_31ens_1ASPTYRS1 - 188
d_32ens_1LMTLMTT
d_33ens_1LMTLMTU
d_34ens_1LMTLMTV
d_35ens_1LMTLMTW
d_36ens_1PTYPTYX
d_41ens_1ASPTYRA1 - 188
d_42ens_1LMTLMTB
d_43ens_1LMTLMTC
d_44ens_1LMTLMTD
d_45ens_1LMTLMTE
d_46ens_1PTYPTYF
d_51ens_1ASPTYRY1 - 188
d_52ens_1LMTLMTZ
d_53ens_1LMTLMTAA
d_54ens_1LMTLMTBA
d_55ens_1LMTLMTCA
d_56ens_1PTYPTYDA
d_61ens_1ASPTYREA1 - 188
d_62ens_1LMTLMTFA
d_63ens_1LMTLMTGA
d_64ens_1LMTLMTHA
d_65ens_1LMTLMTIA
d_66ens_1PTYPTYJA

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Components

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


Mass: 26247.074 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GJB2 / Plasmid: pfastbac / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P29033
#2: Sugar...
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: C24H46O11 / Comment: detergent*YM
#3: Chemical
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 234 / 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: hexameric assembly of human connexin 26 in 90mmHg PCO2, pH7.4, class 2
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 DDM and DTT, was prepared fresh from 10x stock on day of use. The basal buffer was filtered and de-gassed, 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 use. The basal buffer was filtered and de-gassed, and DDM and DTT added. The buffer was pH corrected at point of use to 7.4 using 15% CO2 in 85% N2.
Buffer component
IDConc.NameFormulaBuffer-ID
170 mMsodium chlorideNaCl1
25 %glycerolC3H8O31
31 mMDTTC4H10O2S21
40.03 %DDMC24H46O111
580 mMsodium hydrogen carbonateNaHCO31
61.25 mMsodium dihydrogen phosphateNaH2PO41
73 mMpotassium chlorideKCl1
81 mMmagnesium sulphateMgSO41
94 mMmagnesium chlorideMgCl21
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample monodisperse
Specimen supportDetails: 3ul protein was applied, and blotted for 6 seconds prior to plunge-freeze in atmosphere of 15% CO2/85% N2
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K
Details: 3 microlitres protein applied to grid, blot time 6 seconds, 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: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11647
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionbeta
2EPU2.Ximage acquisitioncollection with aberration-free image shift
4GctfCTF correction
7Coot0.8.9.2 ELmodel fitting
9PHENIX1.18.2-3874model refinement
13RELION3.1-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2655822 / Details: LoG picker implemented in Relion3.1-beta
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218122 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 2ZW3

2zw3

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 73.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00149708
ELECTRON MICROSCOPYf_angle_d0.440813098
ELECTRON MICROSCOPYf_chiral_restr0.03461470
ELECTRON MICROSCOPYf_plane_restr0.00241542
ELECTRON MICROSCOPYf_dihedral_angle_d9.95813414
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2HELECTRON MICROSCOPYNCS constraints0.000704310142532
ens_1d_3HELECTRON MICROSCOPYNCS constraints0.000709680904634
ens_1d_4HELECTRON MICROSCOPYNCS constraints0.000708555196058
ens_1d_5HELECTRON MICROSCOPYNCS constraints0.0800889913693
ens_1d_6HELECTRON MICROSCOPYNCS constraints0.0800890699801

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