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- EMDB-1069: Visualization of the domain structure of an L-type Ca2+ channel u... -

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Basic information

Entry
Database: EMDB / ID: EMD-1069
TitleVisualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy.
Map data3D reconstruction of the skeletal muscle dihydropyridin receptor (DHPR, L-type calcium channel) from single particle images. Low-pass filtered at 23A resolution. Ref.: J Mol Biol. 2003 Sep 5;332(1):171-82 If displayed with chimera (http://www.cgl.ucsf.edu/chimera/download) threshold level for published protein surface (white transparent) = 0.0626 threshold level for published core density (red) = 0.0763
Sample
  • Sample: skeletal muscle dihydropyridine receptor
  • Protein or peptide: DHPR with subunits alpha1, alpha2, beta, gamma, delta
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 21.0 Å
AuthorsWolf M / Eberhart A / Glossmann H / Striessnig J / Grigorieff N
CitationJournal: J Mol Biol / Year: 2003
Title: Visualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy.
Authors: M Wolf / A Eberhart / H Glossmann / J Striessnig / N Grigorieff /
Abstract: The three-dimensional structure of the skeletal muscle voltage-gated L-type calcium channel (Ca(v)1.1; dihydropyridine receptor, DHPR) was determined using electron cryo-microscopy and single- ...The three-dimensional structure of the skeletal muscle voltage-gated L-type calcium channel (Ca(v)1.1; dihydropyridine receptor, DHPR) was determined using electron cryo-microscopy and single-particle averaging. The structure shows a single channel complex with an approximate total molecular mass of 550 kDa, corresponding to the five known subunits of the DHPR, and bound detergent and lipid. Features visible in our structure together with antibody labeling of the beta and alpha(2) subunits allowed us to assign locations for four of the five subunits within the structure. The most striking feature of the structure is the extra-cellular alpha(2) subunit that protrudes from the membrane domain in close proximity to the alpha(1) subunit. The cytosolic beta subunit is located close to the membrane and adjacent to subunits alpha(1), gamma and delta. Our structure correlates well with the functional and biochemical data available for this channel and suggests a three-dimensional model for the excitation-contraction coupling complex consisting of DHPR tetrads and the calcium release channel.
History
DepositionMar 10, 2004-
Header (metadata) releaseMar 11, 2004-
Map releaseMar 11, 2004-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.062
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.062
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1069.gif

Downloads & links

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Map

FileDownload / File: emd_1069.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the skeletal muscle dihydropyridin receptor (DHPR, L-type calcium channel) from single particle images. Low-pass filtered at 23A resolution. Ref.: J Mol Biol. 2003 Sep 5;332(1):171-82 If displayed with chimera (http://www.cgl.ucsf.edu/chimera/download) threshold level for published protein surface (white transparent) = 0.0626 threshold level for published core density (red) = 0.0763
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.39 Å/pix.
x 128 pix.
= 433.92 Å		3.39 Å/pix.
x 128 pix.
= 433.92 Å		3.39 Å/pix.
x 128 pix.
= 433.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.39 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.0148 / Movie #1: 0.062
Minimum - Maximum-0.0164905 - 0.0980307
Average (Standard dev.)0.0013486 (±0.00896609)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 433.92 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.393.393.39
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z433.920433.920433.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0160.0980.001

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Supplemental data

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Sample components

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Entire : skeletal muscle dihydropyridine receptor

EntireName: skeletal muscle dihydropyridine receptor
Components
  • Sample: skeletal muscle dihydropyridine receptor
  • Protein or peptide: DHPR with subunits alpha1, alpha2, beta, gamma, delta

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Supramolecule #1000: skeletal muscle dihydropyridine receptor

SupramoleculeName: skeletal muscle dihydropyridine receptor / type: sample / ID: 1000 / Details: channel complex is solubilized in Digitonin
Oligomeric state: monodisperse channel-detergent complex consisting of 5 subunits
Number unique components: 1
Molecular weightExperimental: 800 KDa / Theoretical: 550 KDa / Method: gel filtration chromatography

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Macromolecule #1: DHPR with subunits alpha1, alpha2, beta, gamma, delta

MacromoleculeName: DHPR with subunits alpha1, alpha2, beta, gamma, delta / type: protein_or_peptide / ID: 1
Name.synonym: DHPR, dihydropyridine receptor, CAv1.1, L type calcium channel
Details: additional MW due to detergent and residual lipid; solubilized in Digitonin
Number of copies: 1
Oligomeric state: monodisperse channel detergent complex with 5 subunits
Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: rabbit skeletal muscle / Cell: animal tissue / Organelle: sarcoplasmatic reticulum / Location in cell: t tubular junction
Molecular weightExperimental: 550 KDa / Theoretical: 800 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.35 mg/mL
BufferpH: 7.4
Details: 50mM Tris-HCl pH7.4, 150mM NaCl, 25uM CaCl2, 1mM Iodoacetamide, 0.1mM Benzamidine, 0.1mM PMSF, 0.1% (w/v) Digitonin.
StainingType: NEGATIVE
Details: 5ul sample on holey carbon grids (Quantifoil) were vitrified by plunging into liqid ethane after blotting with filter paper.
GridDetails: Quantifoil (R) holey carbon copper grids
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 20 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: custom plunger
Method: Blot 5ul sample on negative glow-discharged grids for 5-10sec with Whatman filter paper before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 90 K / Max: 95 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: obj lens astigmatism was corrected at 270,000x magnification
DateFeb 5, 2002
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 180 / Average electron dose: 9 e/Å2
Details: full scan resolution was downsampled by 3x3 pixel averaging.
Od range: 1 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo holder
Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph, CTFFIT3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MRC, IMAGIC, FREALIGN
Details: FREALIGN does not use class averages but performs an orientation search of each individual particle incl. CTF correction. The final map was reconstructed from contributions of 14,056 ...Details: FREALIGN does not use class averages but performs an orientation search of each individual particle incl. CTF correction. The final map was reconstructed from contributions of 14,056 particles and sharpened with a negative temperature factor of 500. The absolute handedness of the reconstruction was not determined.
Number images used: 14056

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