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- PDB-7qer: human Connexin 26 dodecamer at 55mm Hg PCO2, pH7.4 -

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Basic information

Entry
Database: PDB / ID: 7qer
Titlehuman Connexin 26 dodecamer at 55mm Hg 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 / gap junction-mediated intercellular transport / 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 / gap junction channel activity / Gap junction assembly ...Transport of connexons to the plasma membrane / gap junction-mediated intercellular transport / 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 / gap junction channel activity / Gap junction assembly / endoplasmic reticulum-Golgi intermediate compartment / sensory perception of sound / transmembrane transport / cell-cell signaling / calcium ion binding / 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: 2.2 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 1.1May 18, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Oct 23, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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)353,88272
Polymers320,56412
Non-polymers33,31860
Water5,495305
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "H"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "A"
d_9ens_1chain "I"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "L"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLYTYRR1 - 187
d_12ens_1LMTLMTS
d_13ens_1LMTLMTT
d_14ens_1LMTLMTU
d_15ens_1LMTLMTV
d_16ens_1PTYPTYW
d_21ens_1GLYTYRH1 - 187
d_22ens_1LMTLMTI
d_23ens_1LMTLMTJ
d_24ens_1LMTLMTK
d_25ens_1LMTLMTL
d_26ens_1PTYPTYM
d_31ens_1GLYTYRN1 - 187
d_32ens_1LMTLMTO
d_33ens_1LMTLMTP
d_34ens_1LMTLMTQ
d_35ens_1LMTLMTR
d_36ens_1PTYPTYS
d_41ens_1GLYTYRT1 - 187
d_42ens_1LMTLMTU
d_43ens_1LMTLMTV
d_44ens_1LMTLMTW
d_45ens_1LMTLMTX
d_46ens_1PTYPTYY
d_51ens_1GLYTYRZ1 - 187
d_52ens_1LMTLMTA
d_53ens_1LMTLMTB
d_54ens_1LMTLMTC
d_55ens_1LMTLMTD
d_56ens_1PTYPTYE
d_61ens_1GLYTYRF1 - 187
d_62ens_1LMTLMTG
d_63ens_1LMTLMTH
d_64ens_1LMTLMTI
d_65ens_1LMTLMTJ
d_66ens_1PTYPTYK
d_71ens_1GLYTYRL1 - 187
d_72ens_1LMTLMTM
d_73ens_1LMTLMTN
d_74ens_1LMTLMTO
d_75ens_1LMTLMTP
d_76ens_1PTYPTYQ
d_81ens_1GLYTYRA1 - 187
d_82ens_1LMTLMTB
d_83ens_1LMTLMTC
d_84ens_1LMTLMTD
d_85ens_1LMTLMTE
d_86ens_1PTYPTYF
d_91ens_1GLYTYRX1 - 187
d_92ens_1LMTLMTY
d_93ens_1LMTLMTZ
d_94ens_1LMTLMTA
d_95ens_1LMTLMTB
d_96ens_1PTYPTYC
d_101ens_1GLYTYRD1 - 187
d_102ens_1LMTLMTE
d_103ens_1LMTLMTF
d_104ens_1LMTLMTG
d_105ens_1LMTLMTH
d_106ens_1PTYPTYI
d_111ens_1GLYTYRJ1 - 187
d_112ens_1LMTLMTK
d_113ens_1LMTLMTL
d_114ens_1LMTLMTM
d_115ens_1LMTLMTN
d_116ens_1PTYPTYO
d_121ens_1GLYTYRP1 - 187
d_122ens_1LMTLMTQ
d_123ens_1LMTLMTR
d_124ens_1LMTLMTS
d_125ens_1LMTLMTT
d_126ens_1PTYPTYU

NCS oper:
IDCodeMatrixVector
1given(-0.49651050911, -0.868012990822, 0.00554635984782), (-0.867989481216, 0.496414515391, -0.0129185682951), (0.00846019156696, -0.0112283869281, -0.999901169359)122.063059428, 71.8863779859, 153.100342383
2given(0.506450626764, -0.862177526095, 0.0125569162969), (-0.86215904402, -0.506564885961, -0.00859064177806), (0.0137675511477, -0.00647532303753, -0.999884255665)70.9298675782, 126.15371409, 152.676526914
3given(0.999943575793, 0.0105841519544, 0.000906067166801), (0.0105856107697, -0.999942657462, -0.00162068868048), (0.000888861595345, 0.00163018850876, -0.999998276204)-0.73093136041, 108.790521932, 152.369215224
4given(0.492572022162, 0.870236497582, 0.00782567944199), (0.870253177237, -0.492600275301, 0.00209195650665), (0.00567542875098, 0.00577988315167, -0.999967190691)-22.7015087758, 37.5863581295, 151.969024789
5given(-0.49894956477, 0.866628697447, -0.00200812748491), (0.866629350072, 0.49894270716, -0.00312162866792), (-0.00170335242275, -0.00329783748234, -0.999993111406)28.0529141569, -15.997135547, 152.848343642
6given(0.511887482889, -0.859050620169, -0.00179912475256), (0.859049759562, 0.511879430864, 0.00359984101298), (-0.00217151070032, -0.00338825124104, 0.999991902115)71.5914654965, -16.9222343143, 0.370012090733
7given(-0.999995960466, 0.00175501471254, 0.00223583890641), (0.00175938240866, 0.999996545125, 0.00195302639112), (-0.00223240359182, 0.00195695219744, -0.999995593346)100.017176018, -0.212935326257, 152.462093011
8given(0.503416006023, 0.864043595492, -0.000994972394152), (-0.86399407693, 0.503374088309, -0.011347345418), (-0.00930375781243, 0.00657208556454, 0.999935121786)-22.0663305127, 71.2322563331, -0.168917048283
9given(-0.497477686035, 0.867419987567, -0.00992557635897), (-0.867414760126, -0.497546370073, -0.00626446669989), (-0.0103723581152, 0.00549315903842, 0.999931117323)28.5792494672, 125.519538848, -0.0919382702415
10given(-0.999856822885, -0.0088002940495, -0.0144529773682), (0.00867609931922, -0.999925092402, 0.00863335886287), (-0.014527870827, 0.00850672729635, 0.999858278237)101.865170331, 108.107680271, -0.128062544089
11given(-0.488886065374, -0.872344546548, -0.00232533669729), (0.87233378871, -0.488891587172, 0.00433325135038), (-0.00491692573297, 8.99964317895E-5, 0.999987907798)122.671189811, 37.1399951565, 0.16086423772

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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
Source: (gene. exp.) Homo sapiens (human) / Gene: GJB2 / Plasmid: pFastbac
Details (production host): thrombin-cleavable His6 purification tag
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.: 48 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#3: Chemical
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 305 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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: dodecameric assembly of human connexin 26 / Type: ORGANELLE OR CELLULAR COMPONENT / 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 10% CO2 in 90% N2.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
25 %glycerolC3H8O31
31 mMDTTC4H10O2S21
40.03 %DDMC24H46O111
550 mMsodium hydrogen carbonateNaHCO31
61.25 mMsodium dihydrogen phosphateNaH2PO41
73 mMpotassium chlorideKCl1
81 mMmagnesium sulphateMgSO41
92 mMmagnesium chlorideMgCl21
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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 10% CO2/90%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: 81000 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: 5 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4003
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
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
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1679668 / Details: LoG picker implemented in Relion3.1-beta
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 344085 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 2ZW3

2zw3


Accession code: 2ZW3 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 70.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007619248
ELECTRON MICROSCOPYf_angle_d0.602425956
ELECTRON MICROSCOPYf_chiral_restr0.04632928
ELECTRON MICROSCOPYf_plane_restr0.00313072
ELECTRON MICROSCOPYf_dihedral_angle_d17.08426804
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.00071180407128
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.000716214541854
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.000693823599794
ens_1d_5AELECTRON MICROSCOPYNCS constraints0.000708807677831
ens_1d_6AELECTRON MICROSCOPYNCS constraints0.000702324277255
ens_1d_7AELECTRON MICROSCOPYNCS constraints0.00070819885238
ens_1d_8AELECTRON MICROSCOPYNCS constraints0.000707007768095
ens_1d_9AELECTRON MICROSCOPYNCS constraints0.000709415825973
ens_1d_10AELECTRON MICROSCOPYNCS constraints0.000711624665452
ens_1d_11AELECTRON MICROSCOPYNCS constraints0.000712776080575
ens_1d_12AELECTRON MICROSCOPYNCS constraints0.000704095562842

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