Regensburg Center for Biochemistry (RCB), University of Regensburg, Regensburg, Germany.
Methods Mol Biol. 2022;2533:81-96. doi: 10.1007/978-1-0716-2501-9_6.
Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .
最近的技术进步揭示了使用冷冻电镜（cryo-EM）确定大分子复合物高分辨率结构的新前景。在 RNA 聚合酶（Pol）I 研究领域，许多 cryo-EM 研究有助于理解核糖体 RNA 基因转录的高度专业化机制。尽管 cryo-EM 方法本身具有广泛的适用性，但为高分辨率数据收集准备样品可能具有挑战性。在这里，我们描述了用于 Pol I 复合物的纯化和稳定化的策略，特别考虑了该方法的优缺点。我们进一步提供了一种易于实施的方案，用于用自制的碳支撑膜涂覆 EM 网格。总之，我们提出了一种用于 cryo-grid 制备和优化的有效工作流程，包括早期的 cryo-EM 筛选，该筛选可以适应广泛的可溶性样品，以确定高分辨率结构。
