+Open data
-Basic information
Entry | Database: PDB / ID: 6o00 | |||||||||
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Title | apo-LRRC8A in MSP2N2 nanodisc constricted state | |||||||||
Components | Volume-regulated anion channel subunit LRRC8A | |||||||||
Keywords | MEMBRANE PROTEIN / Ion channel / volume-regulation | |||||||||
Function / homology | Function and homology information pre-B cell differentiation / Miscellaneous transport and binding events / volume-sensitive anion channel activity / taurine transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cell volume homeostasis / protein hexamerization ...pre-B cell differentiation / Miscellaneous transport and binding events / volume-sensitive anion channel activity / taurine transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cell volume homeostasis / protein hexamerization / monoatomic anion transport / 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 / identical protein binding / plasma membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.18 Å | |||||||||
Authors | Kern, D.M. / Hite, R.K. / Brohawn, S.G. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6o00.cif.gz | 672.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6o00.ent.gz | 565.7 KB | Display | PDB format |
PDBx/mmJSON format | 6o00.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6o00_validation.pdf.gz | 927.7 KB | Display | wwPDB validaton report |
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Full document | 6o00_full_validation.pdf.gz | 939.5 KB | Display | |
Data in XML | 6o00_validation.xml.gz | 51.7 KB | Display | |
Data in CIF | 6o00_validation.cif.gz | 71.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/o0/6o00 ftp://data.pdbj.org/pub/pdb/validation_reports/o0/6o00 | HTTPS FTP |
-Related structure data
Related structure data | 0564MC 0562C 0563C 6nzwC 6nzzC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | |
EM raw data | EMPIAR-10259 (Title: apo-LRRC8A in MSP2N2 nanodiscs / Data size: 4.6 TB Data #1: apo-LRRC8A in MSP2N2 nanodiscs [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: LRRC8A-DCPIB in MSP1E3D1 nanodisc constricted state / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.565 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Mus musculus (house mouse) | ||||||||||||||||||||
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) | ||||||||||||||||||||
Buffer solution | pH: 7.4 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Details: unspecified | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K / Details: 3 microliter drop size, 3 second blot time |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Sampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement | ||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||
Particle selection | Num. of particles selected: 252655 | ||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||
3D reconstruction | Resolution: 4.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11507 / Symmetry type: POINT |