CRYOELECTRON MICROSCOPY AND IMAGE-ANALYSIS OF THE CARDIAC RYANODINE RECEPTOR

The three-dimensional structure of the cardiac muscle ryanodine recept or (RyR2) is described and compared with its skeletal muscle isoform ( RyR1). Previously, structural studies of RyR2 have not been as informa tive as those for RyR1 because optimal conditions for electron microsc opy, which require low levels of phospholipid, are destabilizing for R yR2. A simple procedure was devised for diluting RyR2 (in phospholipid -containing buffer) into a lipid-free buffer directly on the electron microscope grid, followed by freezing within a few seconds. Cryoelectr on microscopy of RyR2 so prepared yielded images of sufficient quality for analysis by single particle image processing. Averaged projection images for RyR2, as well as for RyR1, prepared under the same conditi ons, were found to be nearly identical in overall dimensions and appea rance at the resolution attained, approximate to 30 Angstrom. An initi al three-dimensional reconstruction of RyR2 was determined (resolution approximate to 41 Angstrom ) and compared with previously reported re constructions of RyR1, Although they looked similar, which is consiste nt with the similarity found for the projection images, and with expec tations based on the 66% amino acid sequence identity of the two isofo rms, structural differences near the corners of the cytoplasmic assemb ly were observed in both two- and three-dimensional studies.