Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM.

ABSTRACT: Ryanodine receptors (RyRs) are large Ca²⁺ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bov

SUBMITTER: Li C 

PROVIDER: S-EPMC11474372 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature