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- EMDB-13230: Cryo-EM structure of homomeric LRRC8A Volume-Regulated Anion Chan... -

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Basic information

Entry
Database: EMDB / ID: EMD-13230
TitleCryo-EM structure of homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
Map datafull-length complex at 3.8 A
Sample
  • Complex: Homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
    • Protein or peptide: synthetic nanobody Sb5
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 speciesMus musculus (house mouse) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDeneka D / Rutz S / Sawicka M
Funding support1 items
OrganizationGrant numberCountry
Swiss National Science Foundation
CitationJournal: Nat Commun / Year: 2021
Title: Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains.
Authors: Dawid Deneka / Sonja Rutz / Cedric A J Hutter / Markus A Seeger / Marta Sawicka / Raimund Dutzler /
Abstract: Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat ...Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.
History
DepositionJul 16, 2021-
Header (metadata) releaseSep 15, 2021-
Map releaseSep 15, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p6k
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
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Supplemental images

emd_13230.png

Downloads & links

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Map

FileDownload / File: emd_13230.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull-length complex at 3.8 A
Voxel sizeX=Y=Z: 1.302 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.04888934 - 0.116420366
Average (Standard dev.)0.00027944145 (±0.0026216889)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 437.47202 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3021.3021.302
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z437.472437.472437.472
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-0.0490.1160.000

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Supplemental data

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Mask #1

Fileemd_13230_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13230_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13230_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Homomeric LRRC8A Volume-Regulated Anion Channel in complex with s...

EntireName: Homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
Components
  • Complex: Homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
    • Protein or peptide: synthetic nanobody Sb5

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Supramolecule #1: Homomeric LRRC8A Volume-Regulated Anion Channel in complex with s...

SupramoleculeName: Homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: Volume-regulated anion channel subunit LRRC8A

MacromoleculeName: Volume-regulated anion channel subunit LRRC8A / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 94.239383 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVTQD KMICLPCKWV TKDSCNDSFR GWAASSPEPT YPNSTVLPT PDTGPTGIKY DLDRHQYNYV DAVCYENRLH WFAKYFPYLV LLHTLIFLAC SNFWFKFPRT SSKLEHFVSI L LKCFDSPW ...String:
MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVTQD KMICLPCKWV TKDSCNDSFR GWAASSPEPT YPNSTVLPT PDTGPTGIKY DLDRHQYNYV DAVCYENRLH WFAKYFPYLV LLHTLIFLAC SNFWFKFPRT SSKLEHFVSI L LKCFDSPW TTRALSETVV EESDPKPAFS KMNGSMDKKS STVSEDVEAT VPMLQRTKSR IEQGIVDRSE TGVLDKKEGE QA KALFEKV KKFRTHVEEG DIVYRLYMRQ TIIKVIKFAL IICYTVYYVH NIKFDVDCTV DIESLTGYRT YRCAHPLATL FKI LASFYI SLVIFYGLIC MYTLWWMLRR SLKKYSFESI REESSYSDIP DVKNDFAFML HLIDQYDPLY SKRFAVFLSE VSEN KLRQL NLNNEWTLDK LRQRLTKNAQ DKLELHLFML SGIPDTVFDL VELEVLKLEL IPDVTIPPSI AQLTGLKELW LYHTA AKIE APALAFLREN LRALHIKFTD IKEIPLWIYS LKTLEELHLT GNLSAENNRY IVIDGLRELK RLKVLRLKSN LSKLPQ VVT DVGVHLQKLS INNEGTKLIV LNSLKKMVNL TELELIRCDL ERIPHSIFSL HNLQEIDLKD NNLKTIEEII SFQHLHR LT CLKLWYNHIA YIPIQIGNLT NLERLYLNRN KIEKIPTQLF YCRKLRYLDL SHNNLTFLPA DIGLLQNLQN LAVTANRI E ALPPELFQCR KLRALHLGNN VLQSLPSRVG ELTNLTQIEL RGNRLECLPV ELGECPLLKR SGLVVEEDLF STLPPEVKE RLWRADKEQA

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Macromolecule #2: synthetic nanobody Sb5

MacromoleculeName: synthetic nanobody Sb5 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.463057 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSSSQVQLVE SGGGLVQAGG SLRLSCAASG FPVAQEIMTW YRQAPGKERE WVAAISSIGD TTAYADSVKG RFTISRDNAK NTVYLQMNS LKPEDTAVYY CAVNVGFTYK GQGTQVTVSA GRAGEQKLIS EEDLNSAVDH HHHHH

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 67.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48917
FSC plot (resolution estimation)

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