Structure of frozen-hydrated triad junctions: a case study in motif searching inside tomograms.

We used tomographic reconstructions of frozen-hydrated triad junctions to determine the structure of the macromolecular complex associated with calcium release from the sarcoplasmic reticulum (SR), during excitation-contraction coupling. Using a rapid motif search algorithm with a reference motif of the ryanodine receptor (RyR) provided by single-particle cryo-electron microscopy, 49 receptors were located in five tomograms. Following co-alignment of the receptors and division into quadrants centered on the 4-fold symmetry axis, the receptors were classified using multivariate statistics. Global and class averages reveal that the SR membrane in the vicinity of the receptor is highly curved, creating an open vestibule with a gap of 4nm between the receptor pore and the calsequestrin layer in the SR lumen. The in-plane densities in the calsequestrin layer have paracrystalline order, consistent with the packing of calsequestrin dimers in the three-dimensional crystal structure. Faint densities ("tethers") extend to the calsequestrin layer from densities in the SR membrane located 15nm from the symmetry axis of the RyR. In a class average of RyRs with proximal transverse tubules (TT), a cytoplasmic density is observed near the receptor that could represent the most consistent location of tethers observed in tomograms between the SR and TT membranes.