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- PDB-8b41: Structure of heteromeric LRRC8A/C (1:1 co-transfected) Volume-Reg... -

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Basic information

Entry
Database: PDB / ID: 8b41
TitleStructure of heteromeric LRRC8A/C (1:1 co-transfected) Volume-Regulated Anion Channel in complex with synthetic nanobody Sb1
Components
  • Synthetic nanobody Sb1
  • Volume-regulated anion channel subunit LRRC8A
  • Volume-regulated anion channel subunit LRRC8C
KeywordsMEMBRANE PROTEIN / Ion channel / Volume-regulated anion channel
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 / cellular response to osmotic stress / monoatomic anion transport ...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 / cellular response to osmotic stress / monoatomic anion transport / cell volume homeostasis / protein hexamerization / response to osmotic stress / monoatomic ion channel complex / fat cell differentiation / intracellular glucose homeostasis / positive regulation of myoblast differentiation / chloride transmembrane transport / positive regulation of insulin secretion / spermatogenesis / lysosomal membrane / endoplasmic reticulum membrane / cell surface / endoplasmic reticulum / signal transduction / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / Leucine Rich Repeat / 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 / Volume-regulated anion channel subunit LRRC8C
Similarity search - Component
Biological speciesMus musculus (house mouse)
Synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSawicka, M. / Dutzler, R.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structure of a volume-regulated heteromeric LRRC8A/C channel.
Authors: Sonja Rutz / Dawid Deneka / Antje Dittmann / Marta Sawicka / Raimund Dutzler /
Abstract: Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions ...Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.
History
DepositionSep 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 28, 2022Group: 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_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Feb 1, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 LRRC8C
F: Volume-regulated anion channel subunit LRRC8C
G: Synthetic nanobody Sb1
H: Synthetic nanobody Sb1
I: Synthetic nanobody Sb1
J: Synthetic nanobody Sb1


Theoretical massNumber of molelcules
Total (without water)619,63310
Polymers619,63310
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29470 Å2
ΔGint-154 kcal/mol
Surface area166970 Å2
MethodPISA

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Components

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


Mass: 95056.312 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8a, Lrrc8 / Production host: Homo sapiens (human) / References: UniProt: Q80WG5
#2: Protein Volume-regulated anion channel subunit LRRC8C / / Factor for adipocyte differentiation 158 / Leucine-rich repeat-containing protein 8C


Mass: 93472.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8c, Ad158, Fad158 / Production host: Homo sapiens (human) / References: UniProt: Q8R502
#3: Antibody
Synthetic nanobody Sb1


Mass: 13115.529 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synthetic construct (others) / Gene: nanobody Sb1 / Production host: E. coli MC1061 (bacteria)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Heteromeric LRRC8A/C Volume-Regulated Anion Channel in complex with synthetic nanobody Sb1COMPLEXall0RECOMBINANT
2Heteromeric LRRC8A/C Volume-Regulated Anion Channel in complexCOMPLEX#1-#21RECOMBINANT
3Synthetic nanobody Sb1COMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33E. coli MC1061 (bacteria)1211845
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329716 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00231992
ELECTRON MICROSCOPYf_angle_d0.44843348
ELECTRON MICROSCOPYf_dihedral_angle_d11.81511817
ELECTRON MICROSCOPYf_chiral_restr0.0394903
ELECTRON MICROSCOPYf_plane_restr0.0035371

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