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- PDB-7qey: human Connexin 26 class 1 hexamer at 90mmHg PCO2, pH7.4 -

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Basic information

Entry
Database: PDB / ID: 7qey
Titlehuman Connexin 26 class 1 hexamer at 90mmHg PCO2, pH7.4
ComponentsGap junction beta-2 protein
KeywordsMEMBRANE PROTEIN / Gap junction / Ion Channel / carbon dioxide sensitive
Function / homologyPHOSPHATIDYLETHANOLAMINE
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 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 1.1Nov 9, 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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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)143,16236
Polymers126,5036
Non-polymers16,65930
Water5,062281
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27320 Å2
ΔGint-21 kcal/mol
Surface area51340 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "H"
d_2ens_1chain "G"
d_3ens_1chain "I"
d_4ens_1chain "J"
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_1ILETYRG1 - 182
d_12ens_1LMTLMTH
d_13ens_1LMTLMTI
d_14ens_1LMTLMTJ
d_15ens_1LMTLMTK
d_16ens_1PTYPTYL
d_21ens_1ILETYRA1 - 182
d_22ens_1LMTLMTB
d_23ens_1LMTLMTC
d_24ens_1LMTLMTD
d_25ens_1LMTLMTE
d_26ens_1PTYPTYF
d_31ens_1ILETYRM1 - 182
d_32ens_1LMTLMTN
d_33ens_1LMTLMTO
d_34ens_1LMTLMTP
d_35ens_1LMTLMTQ
d_36ens_1PTYPTYR
d_41ens_1ILETYRS1 - 182
d_42ens_1LMTLMTT
d_43ens_1LMTLMTU
d_44ens_1LMTLMTV
d_45ens_1LMTLMTW
d_46ens_1PTYPTYX
d_51ens_1ILETYRY1 - 182
d_52ens_1LMTLMTZ
d_53ens_1LMTLMTA
d_54ens_1LMTLMTB
d_55ens_1LMTLMTC
d_56ens_1PTYPTYD
d_61ens_1ILETYRE1 - 182
d_62ens_1LMTLMTF
d_63ens_1LMTLMTG
d_64ens_1LMTLMTH
d_65ens_1LMTLMTI
d_66ens_1PTYPTYJ

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Components

#1: Protein
Gap junction beta-2 protein


Mass: 21083.887 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GJB2 / Plasmid: pfastbac / Production host: Spodoptera frugiperda (fall armyworm)
#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 / Phosphatidylethanolamine


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


Mass: 18.015 Da / Num. of mol.: 281 / 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
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 chlorideNaClSodium chloride1
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-freezing in atmosphere of 15% CO2, 86% 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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
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

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
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 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 414758 / 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: 62.71 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029414
ELECTRON MICROSCOPYf_angle_d0.40812690
ELECTRON MICROSCOPYf_dihedral_angle_d9.5123330
ELECTRON MICROSCOPYf_chiral_restr0.0351422
ELECTRON MICROSCOPYf_plane_restr0.0031500
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2GELECTRON MICROSCOPYNCS constraints0.000709576451878
ens_1d_3GELECTRON MICROSCOPYNCS constraints0.0812309730373
ens_1d_4GELECTRON MICROSCOPYNCS constraints0.0812274660866
ens_1d_5GELECTRON MICROSCOPYNCS constraints0.114914566141
ens_1d_6GELECTRON MICROSCOPYNCS constraints0.000721225850813

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