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

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Basic information

Entry
Database: EMDB / ID: EMD-0563
TitleLRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
Map dataLRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
Sample
  • Complex: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl]oxy}butanoic acid
KeywordsIon channel / volume-regulation / MEMBRANE PROTEIN
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 / monoatomic anion transmembrane transport / taurine 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 / monoatomic anion transmembrane transport / taurine 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsKern DM / Hite RK
Funding support United States, 2 items
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, 2019-
Header (metadata) releaseFeb 27, 2019-
Map releaseFeb 27, 2019-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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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

emd_0563.png

Downloads & links

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Map

FileDownload / File: emd_0563.map.gz / Format: CCP4 / Size: 184 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 10.0 / Movie #1: 10
Minimum - Maximum-35.092934 - 64.369609999999994
Average (Standard dev.)-0.00000000000138 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions364364364
Spacing364364364
CellA=B=C: 396.032 Å
α=β=γ: 90.0 °

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
Components
  • Complex: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl]oxy}butanoic acid

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Supramolecule #1: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state

SupramoleculeName: LRRC8A-DCPIB in MSP1E3D1 nanodisc expanded state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 565 KDa

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Macromolecule #1: Volume-regulated anion channel subunit LRRC8A

MacromoleculeName: Volume-regulated anion channel subunit LRRC8A / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 95.314562 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVTQD KMICLPCKWV TKDSCNDSFR GWAASSPEPT YPNSTVLPT PDTGPTGIKY DLDRHQYNYV DAVCYENRLH WFAKYFPYLV LLHTLIFLAC SNFWFKFPRT SSKLEHFVSI L LKCFDSPW ...String:
MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVTQD KMICLPCKWV TKDSCNDSFR GWAASSPEPT YPNSTVLPT PDTGPTGIKY DLDRHQYNYV DAVCYENRLH WFAKYFPYLV LLHTLIFLAC SNFWFKFPRT SSKLEHFVSI L LKCFDSPW TTRALSETVV EESDPKPAFS KMNGSMDKKS STVSEDVEAT VPMLQRTKSR IEQGIVDRSE TGVLDKKEGE QA KALFEKV KKFRTHVEEG DIVYRLYMRQ TIIKVIKFAL IICYTVYYVH NIKFDVDCTV DIESLTGYRT YRCAHPLATL FKI LASFYI SLVIFYGLIC MYTLWWMLRR SLKKYSFESI REESSYSDIP DVKNDFAFML HLIDQYDPLY SKRFAVFLSE VSEN KLRQL NLNNEWTLDK LRQRLTKNAQ DKLELHLFML SGIPDTVFDL VELEVLKLEL IPDVTIPPSI AQLTGLKELW LYHTA AKIE APALAFLREN LRALHIKFTD IKEIPLWIYS LKTLEELHLT GNLSAENNRY IVIDGLRELK RLKVLRLKSN LSKLPQ VVT DVGVHLQKLS INNEGTKLIV LNSLKKMVNL TELELIRCDL ERIPHSIFSL HNLQEIDLKD NNLKTIEEII SFQHLHR LT CLKLWYNHIA YIPIQIGNLT NLERLYLNRN KIEKIPTQLF YCRKLRYLDL SHNNLTFLPA DIGLLQNLQN LAVTANRI E ALPPELFQCR KLRALHLGNN VLQSLPSRVG ELTNLTQIEL RGNRLECLPV ELGECPLLKR SGLVVEEDLF STLPPEVKE RLWRADKEQA SNSLEVLFQG

UniProtKB: Volume-regulated anion channel subunit LRRC8A

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Macromolecule #2: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 2 / Number of copies: 18 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM

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Macromolecule #3: 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-...

MacromoleculeName: 4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl]oxy}butanoic acid
type: ligand / ID: 3 / Number of copies: 1 / Formula: L9Y
Molecular weightTheoretical: 427.361 Da
Chemical component information

ChemComp-L9Y:
4-{[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl]oxy}butanoic acid

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
20.0 millimolarHEPES
150.0 millimolarPotassium Chloride
1.0 millimolarEDTA
100.0 micromolarDCPIB
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV / Details: 3 microliter drop size, 3 second blot time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 4592 / Average exposure time: 8.0 sec. / Average electron dose: 60.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 752736
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 35435
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)

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