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- EMDB-4361: Cryo-EM density of heteromeric mLRRC8A/C volume-regulated anion c... -

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Basic information

Entry
Database: EMDB / ID: EMD-4361
TitleCryo-EM density of heteromeric mLRRC8A/C volume-regulated anion channel at 7.94 A resolution
Map data
Sample
  • Complex: Heterohexamer of mLRRC8A and mLRRC8C
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.94 Å
AuthorsSawicka M / Deneka D / Lam AKM / Paulino C / Dutzler R
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 10, 2018-
Header (metadata) releaseMay 23, 2018-
Map releaseMay 23, 2018-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.14
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.14
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4361.png

Downloads & links

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Map

FileDownload / File: emd_4361.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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: 0.14 / Movie #1: 0.14
Minimum - Maximum-0.061474808 - 0.32257217
Average (Standard dev.)0.0042789755 (±0.023167463)
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-0.0610.3230.004

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Supplemental data

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Sample components

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Entire : Heterohexamer of mLRRC8A and mLRRC8C

EntireName: Heterohexamer of mLRRC8A and mLRRC8C
Components
  • Complex: Heterohexamer of mLRRC8A and mLRRC8C

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Supramolecule #1: Heterohexamer of mLRRC8A and mLRRC8C

SupramoleculeName: Heterohexamer of mLRRC8A and mLRRC8C / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S

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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

Concentration3.5 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-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2.5 ul sample volume, 3-7 s blotting time.

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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: 1545 / Average exposure time: 12.5 sec. / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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: 101239
CTF correctionSoftware - Name: CTFFIND
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.94 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 27777
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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