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- EMDB-30029: Structure of the human homo-hexameric LRRC8D channel at 4.36 Angstroms -

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Basic information

Entry
Database: EMDB / ID: EMD-30029
TitleStructure of the human homo-hexameric LRRC8D channel at 4.36 Angstroms
Map data
Sample
  • Complex: Hexameric channel of LRC8D_HUMAN
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8D
Function / homology
Function and homology information


Miscellaneous transport and binding events / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cellular response to osmotic stress / protein hexamerization / monoatomic ion channel complex / intracellular glucose homeostasis / plasma membrane => GO:0005886 ...Miscellaneous transport and binding events / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cellular response to osmotic stress / protein hexamerization / monoatomic ion channel complex / intracellular glucose homeostasis / plasma membrane => GO:0005886 / transmembrane transport / membrane => GO:0016020 / endoplasmic reticulum membrane / membrane / plasma membrane / cytoplasm
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / 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 LRRC8D
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsNakamura R / Kasuya G / Yokoyama T / Shirouzu M / Ishitani R / Nureki O
Funding support Japan, 2 items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)16H06294 Japan
Japan Society for the Promotion of Science (JSPS)19K23833 Japan
CitationJournal: Commun Biol / Year: 2020
Title: Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation.
Authors: Ryoki Nakamura / Tomohiro Numata / Go Kasuya / Takeshi Yokoyama / Tomohiro Nishizawa / Tsukasa Kusakizako / Takafumi Kato / Tatsuya Hagino / Naoshi Dohmae / Masato Inoue / Kengo Watanabe / ...Authors: Ryoki Nakamura / Tomohiro Numata / Go Kasuya / Takeshi Yokoyama / Tomohiro Nishizawa / Tsukasa Kusakizako / Takafumi Kato / Tatsuya Hagino / Naoshi Dohmae / Masato Inoue / Kengo Watanabe / Hidenori Ichijo / Masahide Kikkawa / Mikako Shirouzu / Thomas J Jentsch / Ryuichiro Ishitani / Yasunobu Okada / Osamu Nureki /
Abstract: Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at ...Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.
History
DepositionFeb 20, 2020-
Header (metadata) releaseJun 17, 2020-
Map releaseJun 17, 2020-
UpdateJun 17, 2020-
Current statusJun 17, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: PDB-6m04
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30029.png

Downloads & links

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Map

FileDownload / File: emd_30029.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.49 Å
Density
Contour LevelBy AUTHOR: 0.0301 / Movie #1: 0.04
Minimum - Maximum-0.09150517 - 0.21076207
Average (Standard dev.)0.00063587894 (±0.0061800685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 357.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.491.491.49
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z357.600357.600357.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0920.2110.001

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Supplemental data

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Mask #1

Fileemd_30029_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30029_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30029_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Hexameric channel of LRC8D_HUMAN

EntireName: Hexameric channel of LRC8D_HUMAN
Components
  • Complex: Hexameric channel of LRC8D_HUMAN
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8D

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Supramolecule #1: Hexameric channel of LRC8D_HUMAN

SupramoleculeName: Hexameric channel of LRC8D_HUMAN / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Volume-regulated anion channel subunit LRRC8D

MacromoleculeName: Volume-regulated anion channel subunit LRRC8D / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.31118 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MFTLAEVASL NDIQPTYRIL KPWWDVFMDY LAVVMLMVAI FAGTMQLTKD QVVCLPVLPS PVNSKAHTPP GNAEVTTNIP KMEAATNQD QDGRTTNDIS FGTSAVTPDI PLRATYPRTD FALPNQEAKK EKKDPTGRKT NLDFQQYVFI NQMCYHLALP W YSKYFPYL ...String:
MFTLAEVASL NDIQPTYRIL KPWWDVFMDY LAVVMLMVAI FAGTMQLTKD QVVCLPVLPS PVNSKAHTPP GNAEVTTNIP KMEAATNQD QDGRTTNDIS FGTSAVTPDI PLRATYPRTD FALPNQEAKK EKKDPTGRKT NLDFQQYVFI NQMCYHLALP W YSKYFPYL ALIHTIILMV SSNFWFKYPK TCSKVEHFVS ILGKCFESPW TTKALSETAC EDSEENKQRI TGAQTLPKHV ST SSDEGSP SASTPMINKT GFKFSAEKPV IEVPSMTILD KKDGEQAKAL FEKVRKFRAH VEDSDLIYKL YVVQTVIKTA KFI FILCYT ANFVNAISFE HVCKPKVEHL IGYEVFECTH NMAYMLKKLL ISYISIICVY GFICLYTLFW LFRIPLKEYS FEKV REESS FSDIPDVKND FAFLLHMVDQ YDQLYSKRFG VFLSEVSENK LREISLNHEW TFEKLRQHIS RNAQDKQELH LFMLS GVPD AVFDLTDLDV LKLELIPEAK IPAKISQMTN LQELHLCHCP AKVEQTAFSF LRDHLRCLHV KFTDVAEIPA WVYLLK NLR ELYLIGNLNS ENNKMIGLES LRELRHLKIL HVKSNLTKVP SNITDVAPHL TKLVIHNDGT KLLVLNSLKK MMNVAEL EL QNCELERIPH AIFSLSNLQE LDLKSNNIRT IEEIISFQHL KRLTCLKLWH NKIVTIPPSI THVKNLESLY FSNNKLES L PVAVFSLQKL RCLDVSYNNI SMIPIEIGLL QNLQHLHITG NKVDILPKQL FKCIKLRTLN LGQNCITSLP EKVGQLSQL TQLELKGNCF DRLPAQLGQC RMLKKSGLVV EDHLFDTLPL EVKEALNQDI NIPFANGIGT ENLYFQ

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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

Concentration3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
150.0 mMNaClSodium chloridesodium chloride
5.0 mMC4H10O2S2dithiothreitol
0.1 %C56H92O29digitonin

Details: The solution was freshly prepared to avoid digitonin precipitation.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted for 4 seconds before plunging..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 23500
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 79.55 K / Max: 79.55 K
DetailsSpecimen holder is FEI Talos Arctica autogrid holder.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number real images: 3397 / Average exposure time: 15.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: RELION Initial Model (SGD algorithm)
Initial angle assignmentType: RANDOM ASSIGNMENT
Final 3D classificationSoftware - Name: RELION (ver. 2.1)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 2.1) / Details: Maximum likelihood refinement in RELION.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 247154

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6m04:
Structure of the human homo-hexameric LRRC8D channel at 4.36 Angstroms

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