High resolution structure of the membrane embedded skeletal muscle ryanodine receptor

Abstract

The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel, though a recent study resolved the structure with limited resolution in nanodiscs. In the present study we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, a method that improved the incorporation rate by more than 20-fold compared to a dialysis-based approach. In conjunction with new direct-detection cameras, this allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 Å, respectively. This method offers validation for detergent-based structures of the RyR and lays the groundwork for studies utilizing an electrochemical gradient mimicking the native environment, such as that of the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol of the cell at rest.

An optimized approach to study sub-sarcomere structure utilizing super-resolution microscopy with secondary VHH nanobodies

“…We suggest that use of this approach with models of muscle weakness holds potential to help identify potential changes in protein localization and potential disruptions in sub-sarcomere structure that could contribute to functional declines. We note that our approach is not the first to utilize advanced imaging modalities to study skeletal muscle structure (Radermacher et al ., 1994; Blanc et al ., 2000; Soeller & Baddeley, 2013; Glancy et al ., 2014; Sun et al ., 2014; Jayasinghe et al ., 2014, 2015, 2018; Agarwal & Machá, 2016; York & Zheng, 2017; Gemmink et al ., 2018; Yi et al ., 2019; Ghosh et al ., 2019; Szikora et al ., 2020; Daneshparvar et al ., 2020; Oda & Yanagisawa, 2020; Hofemeier et al ., 2021; Rahmanseresht et al ., 2021; van der Pijl et al ., 2021 a ; Wang et al ., 2021; Melville et al ., 2022; Rimoli et al ., 2022; Skorska et al ., 2022; Schueder et al ., 2022; McMillan & Scarff, 2022; Shaib et al , 2022). Many of these approaches implement single molecule super-resolution microscopy techniques, such as stochastic optical reconstruction microscopy (STORM).…”

RYR-1-Related Diseases International Research Workshop: From Mechanisms to Treatments Pittsburgh, PA, U.S.A., 21-22 July 2022

“…Specifically, S48168 (ARM210) binds cooperatively with ATP to the Repeat12 domain to stabilize the RYR1 closed state. In addition to being a powerful tool that complements X-ray crystallography in drug dis-covery, cryo-electron microscopy provides a unique advantage for studying RYR1 in native lipid environments, such as in liposomes [63], which provides an additional avenue for studying the structure and dynamics of RYR1 under a chemical gradient.…”