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- PDB-6djb: Structure of human Volume Regulated Anion Channel composed of SWE... -

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Basic information

Entry
Database: PDB / ID: 6djb
TitleStructure of human Volume Regulated Anion Channel composed of SWELL1 (LRRC8A)
ComponentsVolume-regulated anion channel subunit LRRC8A
KeywordsMEMBRANE PROTEIN / anion channel / LRRC8A / SWELL1
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transport / cell volume homeostasis ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transport / cell volume homeostasis / 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 / signal transduction / membrane / identical protein binding / plasma membrane
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: 4.4 Å
AuthorsKefauver, J.M. / Saotome, K. / Pallesen, J. / Cottrell, C.A. / Ward, A.B. / Patapoutian, A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS083174 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)F31 NS093778 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2018
Title: Structure of the human volume regulated anion channel.
Authors: Jennifer M Kefauver / Kei Saotome / Adrienne E Dubin / Jesper Pallesen / Christopher A Cottrell / Stuart M Cahalan / Zhaozhu Qiu / Gunhee Hong / Christopher S Crowley / Tess Whitwam / Wen- ...Authors: Jennifer M Kefauver / Kei Saotome / Adrienne E Dubin / Jesper Pallesen / Christopher A Cottrell / Stuart M Cahalan / Zhaozhu Qiu / Gunhee Hong / Christopher S Crowley / Tess Whitwam / Wen-Hsin Lee / Andrew B Ward / Ardem Patapoutian /
Abstract: SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with ...SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.
History
DepositionMay 24, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 15, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 22, 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.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)565,9116
Polymers565,9116
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25520 Å2
ΔGint-95 kcal/mol
Surface area192320 Å2

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Components

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


Mass: 94318.516 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): HEK 293F LRRC8(B,C,D,E)-/- / Production host: Homo sapiens (human) / 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: Homomeric Volume Regulated Anion Channel composed of SWELL1
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293F LRRC8(B,C,D,E)-/-
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMsodium chlorideNaClSodium chloride1
30.05 %digitonin1
4EDTA-free protease inhibitor1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4627
Image scansWidth: 3710 / Height: 3838

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Processing

EM software
IDNameVersionCategory
4GctfCTF correction
13RELION2.1b13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 316206
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25719 / Symmetry type: POINT

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