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- EMDB-5048: CLIC2-RyR1 interaction and structural characterization by cryo-el... -

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Basic information

Entry
Database: EMDB / ID: EMD-5048
TitleCLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy
Map dataThis is an image of a surface rendered side view of RyR1-CLIC2 complex
Sample
  • Sample: Skeletal muscle ryanodine receptor and chloride intracellular channel 2 complex
  • Organelle or cellular component: sarcoplasmic reticulum
KeywordsCa2+-release channel / Ca2+ signaling / Chloride intracellular channel 2 / cryo-electron microscopy / ryanodine receptor
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 25.0 Å
AuthorsMeng X / Wang G / Viero C / Wang Q / Mi W / Su X / Wagenknecht T / Williams A / Liu Z / Yin C-C
CitationJournal: J Mol Biol / Year: 2009
Title: CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy.
Authors: Xing Meng / Guoliang Wang / Cedric Viero / Qiongling Wang / Wei Mi / Xiao-Dong Su / Terence Wagenknecht / Alan J Williams / Zheng Liu / Chang-Cheng Yin /
Abstract: Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal ...Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.
History
DepositionJan 20, 2009-
Header (metadata) releaseFeb 9, 2009-
Map releaseApr 15, 2009-
UpdateApr 15, 2009-
Current statusApr 15, 2009Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00029063
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00029063
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5048_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5048.map.gz / Format: CCP4 / Size: 4.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of a surface rendered side view of RyR1-CLIC2 complex
Voxel sizeX=Y=Z: 5.52 Å
Density
Contour Level1: 0.00041 / Movie #1: 0.0002906
Minimum - Maximum-0.000770187 - 0.00310774
Average (Standard dev.)0.0000264358 (±0.00018949)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions105105105
Spacing105105105
CellA=B=C: 579.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.525.525.52
M x/y/z105105105
origin x/y/z0.0000.0000.000
length x/y/z579.600579.600579.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-20-28-19
NX/NY/NZ415638
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS105105105
D min/max/mean-0.0010.0030.000

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Supplemental data

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Sample components

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Entire : Skeletal muscle ryanodine receptor and chloride intracellular cha...

EntireName: Skeletal muscle ryanodine receptor and chloride intracellular channel 2 complex
Components
  • Sample: Skeletal muscle ryanodine receptor and chloride intracellular channel 2 complex
  • Organelle or cellular component: sarcoplasmic reticulum

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Supramolecule #1000: Skeletal muscle ryanodine receptor and chloride intracellular cha...

SupramoleculeName: Skeletal muscle ryanodine receptor and chloride intracellular channel 2 complex
type: sample / ID: 1000
Oligomeric state: Four CLIC2 molecules binds to one homotetramer of RyR1
Number unique components: 2
Molecular weightExperimental: 565 KDa / Theoretical: 565 KDa / Method: Sequencing

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Supramolecule #1: sarcoplasmic reticulum

SupramoleculeName: sarcoplasmic reticulum / type: organelle_or_cellular_component / ID: 1 / Name.synonym: SR / Number of copies: 1 / Oligomeric state: Tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others) / synonym: rabbit / Tissue: skeletal muscle / Organelle: sarcoplasmic reticulum / Location in cell: sarcoplasmic reticulum membrane
Molecular weightExperimental: 2.3 MDa / Theoretical: 2.3 MDa

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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

Concentration0.05 mg/mL
BufferpH: 7.4
Details: 20 mM Na-MOPS, pH7.4, 200 mM NaCl, 2.0 mM EGTA, 0.5% CHAPS, 2 mM DTT, and 2.0 ug/ml leupeptin
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Instrument: OTHER / Details: Vitrification instrument: FEI Vitrobot / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50760 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Oxford CT3500 / Specimen holder model: OTHER / Tilt angle max: 50
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7.0 µm / Number real images: 258 / Average electron dose: 10 e/Å2
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 1
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 15889

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