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- EMDB-4366: Cryo-EM density of homomeric mLRRC8A volume-regulated anion chann... -

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Basic information

Entry
Database: EMDB / ID: EMD-4366
TitleCryo-EM density of homomeric mLRRC8A volume-regulated anion channel at 5.01 A resolution
Map dataNone
Sample
  • Complex: Homohexameric mLRRC8A
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A
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 / protein hexamerization / cell volume homeostasis ...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 / protein hexamerization / cell volume homeostasis / monoatomic anion transport / 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 / identical protein binding / plasma membrane / cytoplasm
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.01 Å
AuthorsSawicka M / Deneka D / Lam AKM / Paulino C / Dutzler R
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_163421 Switzerland
CitationJournal: Nature / Year: 2018
Title: Structure of a volume-regulated anion channel of the LRRC8 family.
Authors: Dawid Deneka / Marta Sawicka / Andy K M Lam / Cristina Paulino / Raimund Dutzler /
Abstract: Volume-regulated anion channels are activated in response to hypotonic stress. These channels are composed of closely related paralogues of the leucine-rich repeat-containing protein 8 (LRRC8) family ...Volume-regulated anion channels are activated in response to hypotonic stress. These channels are composed of closely related paralogues of the leucine-rich repeat-containing protein 8 (LRRC8) family that co-assemble to form hexameric complexes. Here, using cryo-electron microscopy and X-ray crystallography, we determine the structure of a homomeric channel of the obligatory subunit LRRC8A. This protein conducts ions and has properties in common with endogenous heteromeric channels. Its modular structure consists of a transmembrane pore domain followed by a cytoplasmic leucine-rich repeat domain. The transmembrane domain, which is structurally related to connexin proteins, is wide towards the cytoplasm but constricted on the outside by a structural unit that acts as a selectivity filter. An excess of basic residues in the filter and throughout the pore attracts anions by electrostatic interaction. Our work reveals the previously unknown architecture of volume-regulated anion channels and their mechanism of selective anion conduction.
History
DepositionApr 11, 2018-
Header (metadata) releaseMay 16, 2018-
Map releaseMay 16, 2018-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6g9l
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4366.png

Downloads & links

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Map

FileDownload / File: emd_4366.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 322.5 Å		1.08 Å/pix.
x 300 pix.
= 322.5 Å		1.08 Å/pix.
x 300 pix.
= 322.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.075 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.031 / Movie #1: 0.031
Minimum - Maximum-0.014336575 - 0.0882011
Average (Standard dev.)0.00042881264 (±0.0052861706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 322.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0751.0751.075
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z322.500322.500322.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0140.0880.000

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Supplemental data

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Sample components

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Entire : Homohexameric mLRRC8A

EntireName: Homohexameric mLRRC8A
Components
  • Complex: Homohexameric mLRRC8A
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A

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Supramolecule #1: Homohexameric mLRRC8A

SupramoleculeName: Homohexameric mLRRC8A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S-GnTI minus

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.9 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3Tris
250.0 mMNaClSodium chloride
0.12 %C56H92O29Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-50 / Number real images: 9209 / Average exposure time: 12.5 sec. / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37313 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 37313
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 329624
CTF correctionSoftware - Name: CTFFIND
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 54861
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6g9l:
Structure of homomeric mLRRC8A volume-regulated anion channel at 5.01 A resolution

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