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- EMDB-28898: LRRC8A(T48D):C conformation 2 top focus -

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Basic information

Entry
Database: EMDB / ID: EMD-28898
TitleLRRC8A(T48D):C conformation 2 top focus
Map dataLRRC8A(T48D):C conformation 1
Sample
  • Complex: LRRC8A:C
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8C
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water
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 / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cellular response to osmotic stress / protein hexamerization ...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 / cellular response to osmotic stress / protein hexamerization / cell volume homeostasis / monoatomic anion transport / response to osmotic stress / monoatomic ion channel complex / fat cell differentiation / intracellular glucose homeostasis / positive regulation of myoblast differentiation / chloride transmembrane transport / electron transport chain / positive regulation of insulin secretion / spermatogenesis / periplasmic space / electron transfer activity / iron ion binding / lysosomal membrane / heme binding / endoplasmic reticulum membrane / cell surface / endoplasmic reticulum / 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 / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Leucine rich repeat / Leucine-rich repeat, typical subtype ...LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / : / Leucine Rich Repeat / Leucine-rich repeat, SDS22-like subfamily / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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
Soluble cytochrome b562 / 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: 3.15 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_28898.png

Downloads & links

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Map

FileDownload / File: emd_28898.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLRRC8A(T48D):C conformation 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 416 pix.
= 435.968 Å		1.05 Å/pix.
x 416 pix.
= 435.968 Å		1.05 Å/pix.
x 416 pix.
= 435.968 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-1.9281256 - 4.1995807
Average (Standard dev.)0.0029634442 (±0.06019682)
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 1 unsharpened map

Fileemd_28898_additional_1.map
AnnotationLRRC8A(T48D):C conformation 1 unsharpened map
Projections & Slices
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Additional map: LRRC8A(T48D):C conformation 1 composite map

Fileemd_28898_additional_2.map
AnnotationLRRC8A(T48D):C conformation 1 composite map
Projections & Slices
AxesZYX

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Histogram

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Half map: LRRC8A(T48D):C conformation 1

Fileemd_28898_half_map_1.map
AnnotationLRRC8A(T48D):C conformation 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LRRC8A(T48D):C conformation 1

Fileemd_28898_half_map_2.map
AnnotationLRRC8A(T48D):C conformation 1
Projections & Slices
AxesZYX

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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,Soluble cytochrome b562
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8C
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water

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Supramolecule #1: LRRC8A:C

SupramoleculeName: LRRC8A:C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 620 KDa

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Macromolecule #1: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562

MacromoleculeName: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 105.544086 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVDQD KMICLPCKWV TKDSCNDSFR GWAASSADLE DNWETLNDN LKVIEKADNA AQVKDALTKM RAAALDAQKA TPPKLEDKSP DSPEMKDFRH GFDILVGQID DALKLANEGK V KEAQAAAE ...String:
MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVDQD KMICLPCKWV TKDSCNDSFR GWAASSADLE DNWETLNDN LKVIEKADNA AQVKDALTKM RAAALDAQKA TPPKLEDKSP DSPEMKDFRH GFDILVGQID DALKLANEGK V KEAQAAAE QLKTTRNAYI QKYLDTGPTG IKYDLDRHQY NYVDAVCYEN RLHWFAKYFP YLVLLHTLIF LACSNFWFKF PR TSSKLEH FVSILLKCFD SPWTTRALSE TVVEESDPKP AFSKMNGSMD KKSSTVSEDV EATVPMLQRT KSRIEQGIVD RSE TGVLDK KEGEQAKALF EKVKKFRTHV EEGDIVYRLY MRQTIIKVIK FALIICYTVY YVHNIKFDVD CTVDIESLTG YRTY RCAHP LATLFKILAS FYISLVIFYG LICMYTLWWM LRRSLKKYSF ESIREESSYS DIPDVKNDFA FMLHLIDQYD PLYSK RFAV FLSEVSENKL RQLNLNNEWT LDKLRQRLTK NAQDKLELHL FMLSGIPDTV FDLVELEVLK LELIPDVTIP PSIAQL TGL KELWLYHTAA KIEAPALAFL RENLRALHIK FTDIKEIPLW IYSLKTLEEL HLTGNLSAEN NRYIVIDGLR ELKRLKV LR LKSNLSKLPQ VVTDVGVHLQ KLSINNEGTK LIVLNSLKKM VNLTELELIR CDLERIPHSI FSLHNLQEID LKDNNLKT I EEIISFQHLH RLTCLKLWYN HIAYIPIQIG NLTNLERLYL NRNKIEKIPT QLFYCRKLRY LDLSHNNLTF LPADIGLLQ NLQNLAVTAN RIEALPPELF QCRKLRALHL GNNVLQSLPS RVGELTNLTQ IELRGNRLEC LPVELGECPL LKRSGLVVEE DLFSTLPPE VKERLWRADK EQASNSLEVL FQ

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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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Macromolecule #3: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 3 / Number of copies: 17 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71566
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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