一种优化的方案，用于获取和处理与人源 26S 蛋白酶体和 M1-Ub 结合的冷冻电镜数据。
An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub.
机构信息
Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
出版信息
STAR Protoc. 2021 Jan 18;2(1):100278. doi: 10.1016/j.xpro.2020.100278. eCollection 2021 Mar 19.
The 26S proteasome is specialized for regulated protein degradation. It is formed by a regulatory particle (RP) that recognizes ubiquitinated substrates and caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Structural heterogeneity caused by dynamics makes it challenging to observe ubiquitin chains at the RP by cryogenic electron microscopy (cryo-EM). Here, we present a cryo-EM-based protocol we applied to study the human 26S proteasome with ubiquitin chains by using non-cleavable M1-linked hexaubiquitin (M1-Ub) unanchored to a substrate. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).
26S 蛋白酶体专门用于调控蛋白降解。它由一个识别泛素化底物的调节颗粒 (RP) 和一个空心圆柱形核心颗粒 (CP) 组成，底物在 CP 中被蛋白水解。由于动力学引起的结构异质性，使得通过低温电子显微镜 (cryo-EM) 观察 RP 上的泛素链变得具有挑战性。在这里，我们介绍了一种基于 cryo-EM 的方案，我们应用该方案来研究与非切割的 M1 连接的六聚泛素 (M1-Ub) 结合的未锚定在底物上的人 26S 蛋白酶体的泛素链。有关该方案使用和执行的完整详细信息，请参考 Chen 等人 (2020)。
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