TRD1: Dedicated sample preparation for MicroED

TRD 1. MicroED专用样品制备-Eisenberg（电极导线） 总结 用于微晶电子衍射（MicroED）的样品制备是具有挑战性的，并且它是一个限速步骤。 在这里，我们将开发生长纳米晶体的方法，优化它们，并制定出一个协议， 用于MicroED的可再现网格制备。目前，我们使用单颗粒制备用于MicroED的样品， cryoEM方案。这包括将样品溶液移液到EM网格上，印迹过量，并冷冻 通过将网格插入液体乙烷中来检测样品。这一过程对样品来说是苛刻的，特别是印迹法， 会使其暴露在水-空气界面上，损害其结构并破坏下面的晶格。 对于膜蛋白质来说，这个问题会因为蛋白质"软"晶体的生长而加剧， 脂质或去污剂。此外，生长立方相的晶体更具挑战性，因为它们嵌入 进入粘稠的脂质基质，因此几乎不可能在不损坏晶体的情况下进行印迹。 了解哪些结晶和样品制备方法适用于MicroED需要 在选定的生物项目上进行挑战和测试的联合和系统的努力。在此，我们建议 系统地测试纳米晶体生长的条件，并确定哪些程序产生纳米晶体。 最高品质。我们将制定新的战略，保护和发展北极圈。 和保存包。最后，我们将通过一系列的实验来决定， FIB研磨服务于MicroED作为制备生长在立方晶系中的膜蛋白纳米晶体的策略 相位我们将通过三个目标来实现这些目标：1。定向生长和检测; 2. 低温保护策略; 3.用于实验的网格制备MicroED的策略。总的来说， 我们将提供可重复和可靠的样品制备、检测和保存程序， MicroED包括在LCP中生长的膜蛋白。长期目标是使常规和高- 通过MicroED进行结晶和结构测定。

TRD 1. Dedicated sample preparation for MicroED – Eisenberg (Lead) Summary Sample preparation for microcrystal electron diffraction (MicroED) is challenging and it is a rate-limiting step. Here, we will develop methods for growing nanocrystals, optimizing them, and work out a protocol for reproducible grid preparation for MicroED. Currently, we prepare samples for MicroED using single particle cryoEM protocols. This involves pipetting the sample solution onto an EM grid, blotting the excess, and freezing the sample by plunging the grid into liquid ethane. This process is harsh on the sample, particularly that blotting can expose it to the water-air interface compromising its structure and collapsing the underlying crystal lattice. For membrane proteins the problem is exacerbated by the growth of “soft” crystals of protein surrounded by lipids or detergents. Moreover, growing crystals in lipidic cubic phase is even more challenging as they embed into a lipid matrix that is viscous and thus are almost impossible to blot without damaging the crystals. Understanding which crystallization and sample preparation approaches are applicable to MicroED requires a consorted and systematic effort challenged by and tested on select biological projects. Here we propose to systematically test conditions for nanocrystal growth and determine which procedures yield nanocrystals of the highest quality. We will establish new strategies for nanocrystal cryo protection and develop nanocrystal gwoth and preservation kits. Finally, we will decide, through a series of experiments, what are the best practices for FIB milling to serve MicroED as a strategy for preparing nanocrystals of membrane proteins grown in lipidic cubic phase. We will achieve these goals through three aims: 1. Directed nanocrystal growth and detection; 2. Strategies for nanocrystal cryo protection; 3. Strategies for grid preparation MicroED for experiments. Overall, we will deliver reproducible and reliable procedures for sample preparation, detection, and preservation for MicroED including membrane proteins grown in LCP. The long-term goal is to enable routine and high- throughput crystallization and structure determination by MicroED.