3D Mapping of the SPRY2 Domain of RyR1 by Antibody Labeling and Single-Particle cryo-EM

Abstract

The ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) interact functionally at the T tubule/sarcoplasmic reticulum interface of skeletal muscle and are main participants of the excitation-contraction coupling process. The SPRY2 domain of RyR1 has been shown to be relevant for the RyR1/ DHPR interaction. Here, using a combination of immunolabeling and single-particle cryo-EM we have mapped SPRY2 in the 3D structure of RyR1. RyR1 was incubated with three different antibodies against the SPRY2 domain and vitrified for cryo-EM imaging. The two main obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the several possible binding locations of the antibody to the tetrameric RyR1, were overcome by modification of the 3D reconstruction scheme. This enabled us to obtain a 3D reconstruction of RyR1 with the antibody bound, which in turn has allowed us to map the SPRY2 domain in a T-tubule facing domain of RyR1.