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- PDB-6g9l: Structure of homomeric mLRRC8A volume-regulated anion channel at ... -

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Basic information

Entry
Database: PDB / ID: 6g9l
TitleStructure of homomeric mLRRC8A volume-regulated anion channel at 5.01 A resolution
ComponentsVolume-regulated anion channel subunit LRRC8A
KeywordsMEMBRANE PROTEIN / Chloride channel / Swelling-activated / VSOAC / Leucine-rich repeat
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transport / cell volume homeostasis ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transport / cell volume homeostasis / protein hexamerization / response to osmotic stress / monoatomic ion channel complex / intracellular glucose homeostasis / positive regulation of myoblast differentiation / chloride transmembrane transport / positive regulation of insulin secretion / spermatogenesis / lysosomal membrane / cell surface / signal transduction / membrane / identical protein binding / plasma membrane
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / Leucine-rich repeat, SDS22-like subfamily / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Volume-regulated anion channel subunit LRRC8A
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.01 Å
AuthorsSawicka, M. / Deneka, D. / Lam, A.K.M. / Paulino, C. / Dutzler, R.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_163421 Switzerland
CitationJournal: Nature / Year: 2018
Title: Structure of a volume-regulated anion channel of the LRRC8 family.
Authors: Dawid Deneka / Marta Sawicka / Andy K M Lam / Cristina Paulino / Raimund Dutzler /
Abstract: Volume-regulated anion channels are activated in response to hypotonic stress. These channels are composed of closely related paralogues of the leucine-rich repeat-containing protein 8 (LRRC8) family ...Volume-regulated anion channels are activated in response to hypotonic stress. These channels are composed of closely related paralogues of the leucine-rich repeat-containing protein 8 (LRRC8) family that co-assemble to form hexameric complexes. Here, using cryo-electron microscopy and X-ray crystallography, we determine the structure of a homomeric channel of the obligatory subunit LRRC8A. This protein conducts ions and has properties in common with endogenous heteromeric channels. Its modular structure consists of a transmembrane pore domain followed by a cytoplasmic leucine-rich repeat domain. The transmembrane domain, which is structurally related to connexin proteins, is wide towards the cytoplasm but constricted on the outside by a structural unit that acts as a selectivity filter. An excess of basic residues in the filter and throughout the pore attracts anions by electrostatic interaction. Our work reveals the previously unknown architecture of volume-regulated anion channels and their mechanism of selective anion conduction.
History
DepositionApr 11, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 16, 2018Provider: repository / Type: Initial release
Revision 1.1May 30, 2018Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 27, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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  • Deposited structure unit
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  • EMDB-4366
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Volume-regulated anion channel subunit LRRC8A
B: Volume-regulated anion channel subunit LRRC8A
C: Volume-regulated anion channel subunit LRRC8A
D: Volume-regulated anion channel subunit LRRC8A
E: Volume-regulated anion channel subunit LRRC8A
F: Volume-regulated anion channel subunit LRRC8A


Theoretical massNumber of molelcules
Total (without water)565,4366
Polymers565,4366
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25130 Å2
ΔGint-117 kcal/mol
Surface area203750 Å2
MethodPISA

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Components

#1: Protein
Volume-regulated anion channel subunit LRRC8A / / Leucine-rich repeat-containing protein 8A


Mass: 94239.383 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8a, Lrrc8 / Production host: Homo sapiens (human) / References: UniProt: Q80WG5

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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: Homohexameric mLRRC8A / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S-GnTI minus
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisC4H11NO31
2250 mMSodium chlorideNaClSodium chloride1
30.12 %DigitoninC56H92O291
SpecimenConc.: 3.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37313 X / Calibrated magnification: 37313 X / Nominal defocus max: 3200 nm / Nominal defocus min: 500 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12.5 sec. / Electron dose: 1.2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 9209
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13PHENIXmodel refinement
14REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 329624
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 5.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54861 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00472978
ELECTRON MICROSCOPYf_angle_d0.823132528
ELECTRON MICROSCOPYf_dihedral_angle_d10.02629418
ELECTRON MICROSCOPYf_chiral_restr0.0465682
ELECTRON MICROSCOPYf_plane_restr0.00410224

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