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

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Basic information

Entry
Database: PDB / ID: 6m04
TitleStructure of the human homo-hexameric LRRC8D channel at 4.36 Angstroms
ComponentsVolume-regulated anion channel subunit LRRC8D
KeywordsMEMBRANE PROTEIN / Ion channel
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsNakamura, R. / Kasuya, G. / Yokoyama, T. / Shirouzu, M. / Ishitani, R. / Nureki, O.
Funding support Japan, 2items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 17, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Volume-regulated anion channel subunit LRRC8D
B: Volume-regulated anion channel subunit LRRC8D
C: Volume-regulated anion channel subunit LRRC8D
D: Volume-regulated anion channel subunit LRRC8D
E: Volume-regulated anion channel subunit LRRC8D
F: Volume-regulated anion channel subunit LRRC8D


Theoretical massNumber of molelcules
Total (without water)595,8676
Polymers595,8676
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28760 Å2
ΔGint-121 kcal/mol
Surface area199580 Å2

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Components

#1: Protein
Volume-regulated anion channel subunit LRRC8D / / Leucine-rich repeat-containing protein 5 / Leucine-rich repeat-containing protein 8D


Mass: 99311.180 Da / Num. of mol.: 6 / Mutation: L810F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRC8D, LRRC5, UNQ213/PRO239 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / Variant (production host): GntI- / References: UniProt: Q7L1W4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hexameric channel of LRC8D_HUMAN / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S
Buffer solutionpH: 8
Details: The solution was freshly prepared to avoid digitonin precipitation.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2150 mMsodium chlorideNaClSodium chloride1
35 mMdithiothreitolC4H10O2S21
40.1 %digitoninC56H92O291
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotted for 4 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: Specimen holder is FEI Talos Arctica autogrid holder.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 23500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 79.55 K / Temperature (min): 79.55 K
Image recordingAverage exposure time: 15 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3397
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 247154 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00635438
ELECTRON MICROSCOPYf_angle_d1.25648030
ELECTRON MICROSCOPYf_dihedral_angle_d9.26921438
ELECTRON MICROSCOPYf_chiral_restr0.0655620
ELECTRON MICROSCOPYf_plane_restr0.0085960

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