TRD1: Dedicated sample preparation for MicroED

TRD 1.MicroED-Eisenberg(铅)专用样品制备 摘要 微晶电子衍射(MicroED)的样品制备是一个具有挑战性的步骤，也是一个速度限制步骤。 在这里，我们将开发生长纳米晶体的方法，优化它们，并制定出一项协议，以 用于MicroED的可重复栅格准备。目前，我们使用单颗粒来制备微电子能谱样品 冷冻电子显微镜的协议。这包括将样品溶液用吸管移到EM网格上，吸去多余的，然后冷冻 通过将网格插入液态乙烷中，对样品进行采样。这个过程对样品很苛刻，特别是吸墨法 会使其暴露在水-空气界面上，破坏其结构，并使其下面的晶格坍塌。 对于膜蛋白质来说，这个问题因蛋白质晶体的“软”生长而加剧。 脂类或洗涤剂。此外，在脂肪立方相中生长晶体更具挑战性，因为它们嵌入 变成一种粘性的脂质基质，因此几乎不可能在不破坏晶体的情况下吸收。 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受到挑战和测试的协调和系统的努力。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 最好的品质。我们将制定新的纳米晶低温保护策略，发展纳米晶玻璃。 和保存包。最后，我们将通过一系列实验来决定哪些是最佳实践 纤维球磨作为制备脂质立方体中生长的膜蛋白纳米晶的一种策略 相位。我们将通过三个目标来实现这些目标：1.定向纳米晶体的生长和检测； 纳米晶低温保护策略；3.用于实验的栅格制备策略。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- 微电子能谱(MicroED)的结晶和结构测定

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.