2012-10-24 REL 2004-03-10 PDBe PDBe 2004-03-11 2004-03-11 Visualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy. Wolf M, Eberhart A, Glossmann H, Striessnig J, Grigorieff N Wolf M, Eberhart A, Glossmann H, Striessnig J, Grigorieff N Visualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy. J.MOL.BIOL. 332 171 182 2003 12946355 doi:10.1016/S0022-2836(03)00899-4 emd_1069.map.gz Image stored as Reals 128 128 128 -64 -64 -64 63 63 63 128 128 128 433.92 433.92 433.92 90 90 90 X Y Z -0.0164905 0.0980307 0.0013486 0.00896609 1
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3.39 3.39 3.39 0.0148 3D 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 1 skeletal muscle dihydropyridine receptor monodisperse channel-detergent complex consisting of 5 subunits 0.55
channel complex is solubilized in Digitonin
gel filtration chromatography 0.8 protein DHPR with subunits alpha1, alpha2, beta, gamma, delta DHPR, dihydropyridine receptor, CAv1.1, L type calcium channel 0.8 0.55
additional MW due to detergent and residual lipid; solubilized in Digitonin
Oryctolagus cuniculus false monodisperse channel detergent complex with 5 subunits Rabbit 1 animal tissue rabbit skeletal muscle sarcoplasmatic reticulum t tubular junction ETHANE 80 20 HOMEMADE PLUNGER Blot 5ul sample on negative glow-discharged grids for 5-10sec with Whatman filter paper before plunging.
Vitrification instrument: custom plunger
 FEI TECNAI F20 Side entry liquid nitrogen-cooled cryo holder GATAN LIQUID NITROGEN 2002-02-05 200 FLOOD BEAM BRIGHT FIELD 2.0 obj lens astigmatism was corrected at 270,000x magnification 3000 4500 0 0 62000 FIELD EMISSION GUN 9 90 90 95 KODAK SO-163 FILM 180 7 1 8 ZEISS SCAI
full scan resolution was downsampled by 3x3 pixel averaging.
 0.35 5ul sample on holey carbon grids (Quantifoil) were vitrified by plunging into liqid ethane after blotting with filter paper. Quantifoil (R) holey carbon copper grids particle
50mM Tris-HCl pH7.4, 150mM NaCl, 25uM CaCl2, 1mM Iodoacetamide, 0.1mM Benzamidine, 0.1mM PMSF, 0.1% (w/v) Digitonin.
7.4 singleParticle common lines with IMAGIC for initial model, then refinement with FREALIGN MRC, IMAGIC, FREALIGN per micrograph, CTFFIT3 21 FSC 0.5
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.
 14056 C1