title: 3D Mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle cryo-EM.
abstract: The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.
authors: Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Alex
authors: A
authors: Perálvarez-Marín
authors: Hanshen
authors: H
authors: Tae
authors: Philip G
authors: PG
authors: Board
authors: Marco G
authors: MG
authors: Casarotto
authors: Angela F
authors: AF
authors: Dulhunty
authors: Montserrat
authors: M
authors: Samsó
mesh_terms: D000818:Animals; D001666:Binding Sites, Antibody; D020285:Cryoelectron Microscopy; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000276:immunology*
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel