Amino acid residues 4425-4621 localized on the three-dimensional structureof the skeletal muscle ryanodine receptor

We have localized a region contained within the sequence of amino acid resi dues 4425-4621 on the three-dimensional structure of the skeletal muscle ry anodine receptor(RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These imag es, along with images of antibody-free ryanodine receptor, were used to com pute two-dimensional averaged images and three-dimensional reconstructions. :Two-dimensional averages of immunocomplexes in which the ryanodine recept or was in the fourfold symmetrical orientation disclosed four symmetrical r egions of density located on the edges of the receptor's cytoplasmic assemb ly that were absent from control averages of receptor without added antibod y. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is,the first to demonstrate epitope mapping on the three-dimensional struct ure of the ryanodine receptor.