Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment

Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca²⁺ release channels that are responsible for the increase of cytosolic Ca²⁺ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca²⁺ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.