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- EMDB-28895: LRRC8A(T48D):C conformation 2 LRR focus -

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Basic information

Entry
Database: EMDB / ID: EMD-28895
TitleLRRC8A(T48D):C conformation 2 LRR focus
Map dataLRRC8A(T48D):C conformation 2 LRR focus
Sample
  • Complex: LRRC8A:C
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8C
KeywordsION CHANNEL / VOLUME-REGULATION / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsKern DM / Brohawn SG
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM128263 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels.
Authors: David M Kern / Julia Bleier / Somnath Mukherjee / Jennifer M Hill / Anthony A Kossiakoff / Ehud Y Isacoff / Stephen G Brohawn /
Abstract: Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate ...Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (SWELL1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents across cell types. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here we leverage a fiducial-tagging strategy to determine single-particle cryo-EM structures of heterohexameric LRRC8A:C channels in multiple conformations. Compared to homomers, LRRC8A:C channels show pronounced differences in architecture due to heterotypic LRR interactions that displace subunits away from the conduction axis and poise the channel for activation. Structures and functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. These results provide insight into determinants for heteromeric LRRC8 channel assembly, activity and gating by lipids.
History
DepositionNov 18, 2022-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28895.png

Downloads & links

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Map

FileDownload / File: emd_28895.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLRRC8A(T48D):C conformation 2 LRR focus
Voxel sizeX=Y=Z: 1.048 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.2133472 - 2.9784782
Average (Standard dev.)0.002790026 (±0.044170972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 435.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: LRRC8A(T48D):C conformation 2 LRR focus unsharpened

Fileemd_28895_additional_1.map
AnnotationLRRC8A(T48D):C conformation 2 LRR focus unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LRRC8A(T48D):C conformation 2 LRR focus

Fileemd_28895_half_map_1.map
AnnotationLRRC8A(T48D):C conformation 2 LRR focus
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LRRC8A(T48D):C conformation 2 LRR focus

Fileemd_28895_half_map_2.map
AnnotationLRRC8A(T48D):C conformation 2 LRR focus
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : LRRC8A:C

EntireName: LRRC8A:C
Components
  • Complex: LRRC8A:C
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8C

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Supramolecule #1: LRRC8A:C

SupramoleculeName: LRRC8A:C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 620 KDa

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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: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 85.002633 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DTGPTGIKYD LDRHQYNYVD AVCYENRLHW FAKYFPYLVL LHTLIFLACS NFWFKFPRTS SKLEHFVSIL LKCFDSPWTT RALSETVVE ESDPKPAFSK MNGSMDKKSS TVSEDVEATV PMLQRTKSRI EQGIVDRSET GVLDKKEGEQ AKALFEKVKK F RTHVEEGD ...String:
DTGPTGIKYD LDRHQYNYVD AVCYENRLHW FAKYFPYLVL LHTLIFLACS NFWFKFPRTS SKLEHFVSIL LKCFDSPWTT RALSETVVE ESDPKPAFSK MNGSMDKKSS TVSEDVEATV PMLQRTKSRI EQGIVDRSET GVLDKKEGEQ AKALFEKVKK F RTHVEEGD IVYRLYMRQT IIKVIKFALI ICYTVYYVHN IKFDVDCTVD IESLTGYRTY RCAHPLATLF KILASFYISL VI FYGLICM YTLWWMLRRS LKKYSFESIR EESSYSDIPD VKNDFAFMLH LIDQYDPLYS KRFAVFLSEV SENKLRQLNL NNE WTLDKL RQRLTKNAQD KLELHLFMLS GIPDTVFDLV ELEVLKLELI PDVTIPPSIA QLTGLKELWL YHTAAKIEAP ALAF LRENL RALHIKFTDI KEIPLWIYSL KTLEELHLTG NLSAENNRYI VIDGLRELKR LKVLRLKSNL SKLPQVVTDV GVHLQ KLSI NNEGTKLIVL NSLKKMVNLT ELELIRCDLE RIPHSIFSLH NLQEIDLKDN NLKTIEEIIS FQHLHRLTCL KLWYNH IAY IPIQIGNLTN LERLYLNRNK IEKIPTQLFY CRKLRYLDLS HNNLTFLPAD IGLLQNLQNL AVTANRIEAL PPELFQC RK LRALHLGNNV LQSLPSRVGE LTNLTQIELR GNRLECLPVE LGECPLLKRS GLVVEEDLFS TLPPEVKERL WRADKEQA S NSLEVLFQ

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Macromolecule #2: Volume-regulated anion channel subunit LRRC8C

MacromoleculeName: Volume-regulated anion channel subunit LRRC8C / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 93.624594 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MIPVTEFRQF SEQQPAFRVL KPWWDVFTDY LSVAMLMIGV FGCTLQVMQD KIICLPKRVQ PAQNHSSVPN VSQAVISTTP LPPPKPSPT NPATVEMKGL KTDLDLQQYS FINQMCYERA LHWYAKYFPY LVLIHTLVFM LCSNFWFKFP GSSSKIEHFI S ILGKCFDS ...String:
MIPVTEFRQF SEQQPAFRVL KPWWDVFTDY LSVAMLMIGV FGCTLQVMQD KIICLPKRVQ PAQNHSSVPN VSQAVISTTP LPPPKPSPT NPATVEMKGL KTDLDLQQYS FINQMCYERA LHWYAKYFPY LVLIHTLVFM LCSNFWFKFP GSSSKIEHFI S ILGKCFDS PWTTRALSEV SGEDSEEKDN RKNNMNRSGT IQSGPEGNLV RSQSLKSIPE KFVVDKSAAG ALDKKEGEQA KA LFEKVKK FRLHVEEGDI LYAMYVRQTV LKVIKFLIII AYNSALVSKV QFTVDCNVDI QDMTGYKNFS CNHTMAHLFS KLS FCYLCF VSIYGLTCLY TLYWLFYRSL REYSFEYVRQ ETGIDDIPDV KNDFAFMLHM IDQYDPLYSK RFAVFLSEVS ENKL KQLNL NNEWTPDKLR QKLQTNAHNR LELPLIMLSG LPDTVFEITE LQSLKLEIIK NVMIPATIAQ LDNLQELCLH QCSVK IHSA ALSFLKENLK VLSVKFDDMR ELPPWMYGLR NLEELYLVGS LSHDISKNVT LESLRDLKSL KILSIKSNVS KIPQAV VDV SSHLQKMCVH NDGTKLVMLN NLKKMTNLTE LELVHCDLER IPHAVFSLLS LQELDLKENN LKSIEEIVSF QHLRKLT VL KLWYNSIAYI PEHIKKLTSL ERLFFSHNKV EVLPSHLFLC NKIRYLDLSY NDIRFIPPEI GVLQSLQYFS ITCNKVES L PDELYFCKKL KTLKIGKNSL SVLSPKIGNL LFLSYLDIKG NHFEVLPPEL GDCRALKRAG LVVEDALFET LPSDVREQM KADSNSENLY FQG

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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

Concentration2 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.002 µm / Nominal defocus min: 0.0006 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71198

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