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- PDB-6o00: apo-LRRC8A in MSP2N2 nanodisc constricted state -

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Basic information

Entry
Database: PDB / ID: 6o00
Titleapo-LRRC8A in MSP2N2 nanodisc constricted state
ComponentsVolume-regulated anion channel subunit LRRC8A
KeywordsMEMBRANE PROTEIN / Ion channel / volume-regulation
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / taurine transmembrane transport / monoatomic anion transmembrane transport / protein hexamerization / cell volume homeostasis ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / taurine transmembrane transport / monoatomic anion transmembrane transport / protein hexamerization / cell volume homeostasis / monoatomic anion transport / response to osmotic stress / intracellular glucose homeostasis / monoatomic ion channel complex / positive regulation of myoblast differentiation / chloride transmembrane transport / positive regulation of insulin secretion / spermatogenesis / lysosomal membrane / cell surface / identical protein binding / plasma membrane
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / : / : / Leucine-rich repeat region / 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. ...LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / : / : / Leucine-rich repeat region / 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: 4.18 Å
AuthorsKern, D.M. / Hite, R.K. / Brohawn, S.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM123496 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM128263 United States
CitationJournal: Elife / Year: 2019
Title: Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs.
Authors: David M Kern / SeCheol Oh / Richard K Hite / Stephen G Brohawn /
Abstract: Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in ...Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here, we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.
History
DepositionFeb 14, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 27, 2019Provider: repository / Type: Initial release
Revision 1.0Feb 27, 2019Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Feb 27, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 27, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 27, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 27, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 27, 2019Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 27, 2019Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Mar 13, 2019Group: Data collection / Database references
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Revision 1.2Nov 27, 2019Group: Database references / Category: pdbx_database_related / Item: _pdbx_database_related.db_id
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
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] / _pdbx_audit_support.funding_organization
Revision 1.4Nov 13, 2024Group: Data collection / Database references / Structure summary
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Revision 1.5May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
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Structure visualization

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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)571,8876
Polymers571,8876
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 95314.562 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q80WG5
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: LRRC8A-DCPIB in MSP1E3D1 nanodisc constricted state / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.565 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMPotassium Chloride1
31 mMEDTA1
4100 micromolarDCPIB1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K / Details: 3 microliter drop size, 3 second blot time

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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 defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 70.3 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1786
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
8PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 252655
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 4.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11507 / Symmetry type: POINT

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