title: Structure of glutaraldehyde cross-linked ryanodine receptor.
abstract: The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [(3)H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.
authors: Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
authors: Joshua D
authors: JD
authors: Strauss
authors: Terence
authors: T
authors: Wagenknecht
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral / Q000737:chemistry*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D005976:Glutaral