Cryo-EM Studies of RyR1 Channel in Detergent-Free Aqueous Environment

Abstract

Skeletal muscle ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum membrane and plays a key role in excitation-contraction coupling. Obtaining high-resolution 3D structure of RyR1 is a formidable challenge due to its enormous size (∼2.3 MDa), dynamic nature and location in the lipid membrane in native state. Detergents are traditionally used to make membrane proteins water soluble and suitable for single-particle cryo-EM. However, detergents tend to destabilize and inactivate membrane proteins. In addition, the presence of detergent in the protein sample reduces surface tension of water making it difficult to control the ice thickness and distribution of channel particles in cryospecimen, and leads to low-contrast in cryo-images. To overcome these difficulties we used amphipathic polymer, amphipol 8-35 (APol8-35), to substitute for detergent in RyR1 preparations. We tested functionality of RyR1/APol8-35 in a [3H]ryanodine binding assay, which yielded Kd and Bmax values similar to those of the purified RyR1 in the presence of CHAPS, indicating that the high-affinity binding site for ryanodine is retained in RyR1/APol8-35. The use of Apol8-35 allowed us to reproducibly obtain ice-embedded specimens of RyR1 for cryo-EM analysis and resulted in improved ice thickness with channel particles uniformly distributed across the holes in the grids. The protein contrast of ice-embedded RyR1/APol8-35 is substantially higher than in detergent preparations using a traditional CCD detector. Furthermore, low-dose images of vitrified RyR1/APol8-35 have been collected on the DE-12 back-thinned DDD camera using JEM3200FSC electron cryomicrosope operated at 300 keV at liquid nitrogen temperature. Preliminary analysis of these cryo-EM images demonstrates image signals that extend beyond ∼6A. Optimal conditions for both sample vitrification and data acquisition were reached in order to achieve a higher resolution RyR1 structure. Supported by NIH (R21AR063255, R01GM072804, R01GM080139, P41GM103832) and AHA (12GRNT10510002).