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- PDB-6nzw: LRRC8A-DCPIB in MSP1E3D1 nanodisc constricted state -

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Basic information

Entry
Database: PDB / ID: 6nzw
TitleLRRC8A-DCPIB in MSP1E3D1 nanodisc constricted state
ComponentsVolume-regulated anion channel subunit LRRC8A
KeywordsMEMBRANE PROTEIN / Ion channel / volume-regulation
Function / homologyLeucine-rich repeat, typical subtype / Miscellaneous transport and binding events / Leucine-rich repeat profile. / Leucine rich repeat / Pannexin-like TM region of LRRC8 / LRRC8 / Leucine-rich repeat domain superfamily / LRRC8, pannexin-like TM region / Leucine-rich repeat / pre-B cell differentiation ...Leucine-rich repeat, typical subtype / Miscellaneous transport and binding events / Leucine-rich repeat profile. / Leucine rich repeat / Pannexin-like TM region of LRRC8 / LRRC8 / Leucine-rich repeat domain superfamily / LRRC8, pannexin-like TM region / Leucine-rich repeat / pre-B cell differentiation / taurine transport / volume-sensitive anion channel activity / aspartate transmembrane transport / anion transmembrane transport / anion transport / cell volume homeostasis / ion channel complex / protein hexamerization / response to osmotic stress / integral component of plasma membrane / cell surface / identical protein binding / plasma membrane / cytoplasm / Volume-regulated anion channel subunit LRRC8A
Function and homology information
Specimen sourceMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.21 Å resolution
AuthorsKern, D.M. / Hite, R.K. / Brohawn, S.G.
Funding supportUnited States , 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesDP2GM123496United States
National Institutes of Health/National Institute of General Medical SciencesF32GM128263United 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2019 / Release: Feb 27, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Feb 27, 2019Structure modelrepositoryInitial release
1.1Mar 13, 2019Structure modelData collection / Database referencescitation / citation_author / em_admin / pdbx_database_proc_citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)585,99625
Polyers571,8876
Non-polymers14,10919
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 95314.562 Da / Num. of mol.: 6 / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8a, Lrrc8 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q80WG5
#2: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 18 / Formula: C42H82NO8P / POPC
#3: Chemical ChemComp-L9Y / 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl]oxy}butanoic acid


Mass: 427.361 Da / Num. of mol.: 1 / Formula: C22H28Cl2O4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: LRRC8A-DCPIB in MSP1E3D1 nanodisc constricted state / Type: COMPLEX / Entity ID: 1 / 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 supportGrid material: GOLD / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 kelvins / 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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 60.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 4592
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 microns / Width: 7420 / Height: 7676 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.14_3211: / Classification: refinement
EM software
IDNameVersionCategory
10RELION3.0initial Euler assignment
11RELION3.0final Euler assignment
13RELION3.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 752736
SymmetryPoint symmetry: C6
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 25153 / Symmetry type: POINT

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