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- EMDB-6952: Structure of the human homo-hexameric LRRC8A channel at 4.25 Angstroms -

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Basic information

Entry
Database: EMDB / ID: EMD-6952
TitleStructure of the human homo-hexameric LRRC8A channel at 4.25 Angstroms
Map data
Sample
  • Complex: Hexameric channel of LRC8A_HUMAN
    • 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 / monoatomic anion 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 / aspartate transmembrane transport / monoatomic anion 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 speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsKasuya G / Nakane T / Yokoyama T / Shirouzu M / Ishitani R / Nureki O
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Cryo-EM structures of the human volume-regulated anion channel LRRC8.
Authors: Go Kasuya / Takanori Nakane / Takeshi Yokoyama / Yanyan Jia / Masato Inoue / Kengo Watanabe / Ryoki Nakamura / Tomohiro Nishizawa / Tsukasa Kusakizako / Akihisa Tsutsumi / Haruaki Yanagisawa ...Authors: Go Kasuya / Takanori Nakane / Takeshi Yokoyama / Yanyan Jia / Masato Inoue / Kengo Watanabe / Ryoki Nakamura / Tomohiro Nishizawa / Tsukasa Kusakizako / Akihisa Tsutsumi / Haruaki Yanagisawa / Naoshi Dohmae / Motoyuki Hattori / Hidenori Ichijo / Zhiqiang Yan / Masahide Kikkawa / Mikako Shirouzu / Ryuichiro Ishitani / Osamu Nureki /
Abstract: Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell ...Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.
History
DepositionApr 29, 2018-
Header (metadata) releaseAug 15, 2018-
Map releaseAug 15, 2018-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045391
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045391
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5zsu
  • Surface level: 0.045391
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6952.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.49 Å
Density
Contour LevelBy AUTHOR: 0.045391 / Movie #1: 0.045391
Minimum - Maximum-0.13621354 - 0.22768216
Average (Standard dev.)0.00058447453 (±0.008339007)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 357.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.491.491.49
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z357.600357.600357.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1360.2280.001

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Supplemental data

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Mask #1

Fileemd_6952_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_6952_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_6952_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Hexameric channel of LRC8A_HUMAN

EntireName: Hexameric channel of LRC8A_HUMAN
Components
  • Complex: Hexameric channel of LRC8A_HUMAN
    • Protein or peptide: Volume-regulated anion channel subunit LRRC8A

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Supramolecule #1: Hexameric channel of LRC8A_HUMAN

SupramoleculeName: Hexameric channel of LRC8A_HUMAN / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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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: Homo sapiens (human)
Molecular weightTheoretical: 95.271516 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVTQD KMICLPCKWV TKDSCNDSFR GWAAPGPEPT YPNSTILPT PDTGPTGIKY DLDRHQYNYV DAVCYENRLH WFAKYFPYLV LLHTLIFLAC SNFWFKFPRT SSKLEHFVSI L LKCFDSPW ...String:
MIPVTELRYF ADTQPAYRIL KPWWDVFTDY ISIVMLMIAV FGGTLQVTQD KMICLPCKWV TKDSCNDSFR GWAAPGPEPT YPNSTILPT PDTGPTGIKY DLDRHQYNYV DAVCYENRLH WFAKYFPYLV LLHTLIFLAC SNFWFKFPRT SSKLEHFVSI L LKCFDSPW TTRALSETVV EESDPKPAFS KMNGSMDKKS STVSEDVEAT VPMLQRTKSR IEQGIVDRSE TGVLDKKEGE QA KALFEKV KKFRTHVEEG DIVYRLYMRQ TIIKVIKFIL 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 LNSLKKMANL TELELIRCDL ERIPHSIFSL HNLQEIDLKD NNLKTIEEII SFQHLHR LT CLKLWYNHIA YIPIQIGNLT NLERLYLNRN KIEKIPTQLF YCRKLRYLDL SHNNLTFLPA DIGLLQNLQN LAITANRI E TLPPELFQCR KLRALHLGNN VLQSLPSRVG ELTNLTQIEL RGNRLECLPV ELGECPLLKR SGLVVEEDLF NTLPPEVKE RLWRADKEQA GTENLYFQ

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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

Concentration15.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
150.0 mMNaClSodium chloridesodium chloride
5.0 mMC4H10O2S2dithiothreitol
0.1 %C56H92O29digitonin

Details: Solution was freshly prepared to avoid digitonin precipitation.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 53.329 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted for 12 seconds before plunging..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 23500
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 79.55 K / Max: 79.55 K
DetailsSpecimen holder is FEI Talos Arctica autogrid holder.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 3 / Number real images: 5305 / Average exposure time: 15.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: RELION Initial Model (SGD algorithm)
Initial angle assignmentType: RANDOM ASSIGNMENT
Final 3D classificationSoftware - Name: RELION (ver. 2.1)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 2.1) / Details: Maximum likelihood refinement in RELION.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 164749
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-5zsu:
Structure of the human homo-hexameric LRRC8A channel at 4.25 Angstroms

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