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- EMDB-0563: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state -

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Entry
Database: EMDB / ID: 0563
TitleLRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
Map dataLRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
SampleLRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
  • Volume-regulated anion channel subunit LRRC8A
  • (ligand) x 2
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
SourceMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / 3.32 Å resolution
AuthorsKern DM / Hite RK / Brohawn SG
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 ReportPDB-ID: 6nzz

SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2019 / Header (metadata) release: Feb 27, 2019 / Map release: Feb 27, 2019 / Last update: Mar 13, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nzz
  • Surface level: 10
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0563.map.gz (map file in CCP4 format, 192915 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
364 pix
1.09 Å/pix.
= 396.032 Å
364 pix
1.09 Å/pix.
= 396.032 Å
364 pix
1.09 Å/pix.
= 396.032 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.088 Å
Density
Contour Level:10.0 (by author), 10 (movie #1):
Minimum - Maximum-35.092934 - 64.369609999999994
Average (Standard dev.)-0.00000000000138 (1.0)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderZYX
Dimensions364364364
Origin0.00.00.0
Limit363.0363.0363.0
Spacing364364364
CellA=B=C: 396.032 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0881.0881.088
M x/y/z364364364
origin x/y/z0.0000.0000.000
length x/y/z396.032396.032396.032
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ364364364
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS364364364
D min/max/mean-35.09364.370-0.000

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Supplemental data

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Sample components

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Entire LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state

EntireName: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state / Number of components: 4

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Component #1: protein, LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state

ProteinName: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state / Recombinant expression: No
MassTheoretical: 565 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #2: protein, Volume-regulated anion channel subunit LRRC8A

ProteinName: Volume-regulated anion channel subunit LRRC8A / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 95.314562 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: ligand, (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propy...

LigandName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
Number of Copies: 18 / Recombinant expression: No
MassTheoretical: 0.760076 kDa

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Component #4: ligand, 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dih...

LigandName: 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl]oxy}butanoic acid
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.427361 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.8 mg/ml / pH: 7.4
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 295.15 K / Humidity: 100 % / 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
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60.8 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200.0 - 2500.0 nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4592 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C6 (6 fold cyclic) / Number of projections: 35435
3D reconstructionSoftware: RELION / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF

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