Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
J Struct Biol. 2013 Mar;181(3):300-6. doi: 10.1016/j.jsb.2013.01.001. Epub 2013 Jan 17.
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.
兰尼碱受体（RyR）家族的钙释放通道在兴奋-收缩偶联（ECC）中起着至关重要的作用。与二氢吡啶受体（DHPR）、钙网蛋白和其他几个较小的调节和衔接蛋白一起，RyR 形成一个被称为 ECC 机械的大型动态复合物。在这里，我们描述了一种简单的交联程序，可用于稳定 ECC 机械中脆弱的成分，目的是通过单颗粒冷冻电子显微镜（cryo-EM）进行结构阐明。作为模型系统，使用 FK506 结合蛋白（FKBP12）和 RyR1 的复合物来测试交联方案。戊二醛固定导致受体结合的 FKBP12 与 RyR1 完全交联，并且还导致构成 RyR 的四个亚基彼此之间广泛交联，而不会损害 RyR1 的超微结构。通过单颗粒 cryo-EM 在 2D 平均值中可视化 FKBP12 与 RyR1 的交联。对照 RyR1 和交联 RyR1 的 3D 重建的比较显示跨膜组装和细胞质区域的微小构象变化。亚基间交联增强了 [(3)H]ryanodine 与 RyR1 的结合。基于我们的发现，我们提出戊二醛的 RyR1 亚基间交联诱导 RyR1 采用开放样构象。
