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- PDB-5zsu: Structure of the human homo-hexameric LRRC8A channel at 4.25 Angstroms -

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Basic information

Entry
Database: PDB / ID: 5zsu
TitleStructure of the human homo-hexameric LRRC8A channel at 4.25 Angstroms
ComponentsVolume-regulated anion channel subunit LRRC8A
KeywordsMEMBRANE PROTEIN
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cell volume homeostasis / monoatomic anion transport ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cell volume homeostasis / monoatomic anion transport / protein hexamerization / 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 / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / 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 speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsKasuya, G. / Nakane, T. / Yokoyama, T. / Shirouzu, M. / Ishitani, R. / Nureki, O.
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Cryo-EM structures of the human volume-regulated anion channel LRRC8.
Authors: Go Kasuya / Takanori Nakane / Takeshi Yokoyama / Yanyan Jia / Masato Inoue / Kengo Watanabe / Ryoki Nakamura / Tomohiro Nishizawa / Tsukasa Kusakizako / Akihisa Tsutsumi / Haruaki Yanagisawa ...Authors: Go Kasuya / Takanori Nakane / Takeshi Yokoyama / Yanyan Jia / Masato Inoue / Kengo Watanabe / Ryoki Nakamura / Tomohiro Nishizawa / Tsukasa Kusakizako / Akihisa Tsutsumi / Haruaki Yanagisawa / Naoshi Dohmae / Motoyuki Hattori / Hidenori Ichijo / Zhiqiang Yan / Masahide Kikkawa / Mikako Shirouzu / Ryuichiro Ishitani / Osamu Nureki /
Abstract: Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell ...Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.
History
DepositionApr 29, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 15, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 22, 2018Group: Data collection / Structure summary / Category: struct / Item: _struct.title
Revision 1.2Sep 5, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Sep 26, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Structure visualization

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Assembly

Deposited unit
A: Volume-regulated anion channel subunit LRRC8A
B: Volume-regulated anion channel subunit LRRC8A
C: Volume-regulated anion channel subunit LRRC8A
D: Volume-regulated anion channel subunit LRRC8A
E: Volume-regulated anion channel subunit LRRC8A
F: Volume-regulated anion channel subunit LRRC8A


Theoretical massNumber of molelcules
Total (without water)571,6296
Polymers571,6296
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24510 Å2
ΔGint-70 kcal/mol
Surface area203950 Å2

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Components

#1: Protein
Volume-regulated anion channel subunit LRRC8A / Leucine-rich repeat-containing protein 8A / Swelling protein 1


Mass: 95271.516 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRRC8A, KIAA1437, LRRC8, SWELL1, UNQ221/PRO247 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / Tissue (production host): Embryonic kidney / References: UniProt: Q8IWT6

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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 LRC8A_HUMAN / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Details: Solution was freshly prepared to avoid digitonin precipitation.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2150 mMsodium chlorideNaCl1
35 mMdithiothreitolC4H10O2S21
40.1 %digitoninC56H92O291
SpecimenConc.: 15 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 12 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 FIELD / Nominal magnification: 23500 X / Nominal defocus max: 3500 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 grids imaged: 3 / Num. of real images: 5305
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
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: C3 (3 fold cyclic)
3D reconstructionResolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164749 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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