TRD1: Dedicated sample preparation for MicroED

TRD1：MicroED 专用样品制备

• 批准号: 10460922
• 负责人: DAVID EISENBERG
• 金额: $ 21.08万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10460922
• 关键词: Adopted; Air; Alcohols; Biological; Biological Assay; Cryoelectron Microscopy; Cryopreservation; Crystallization; Detection; Detergents; Diffusion; Electron Diffraction Microscopy; Electron Microscope; Ethane; Foundations; Freezing; G-Protein-Coupled Receptors; Goals; Growth; Ice; Image; Investigation; Ions; Lead; Lipase; Lipids; Liquid substance; Membrane Proteins; Methodology; Methods; Modality; Phase; Polishes; Preparation; Procedures; Process; Proteins; Protocols documentation; Reproducibility; Sampling; Series; Solvents; Specific qualifier value; Structure; Techniques; Testing; Thick; Violence; Viscosity; Water; Work; density; electron diffraction; experimental study; imaging facilities; improved; nanocrystal; nanometer; nanoscale; particle; preservation; vapor

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.

TRD 1. MicroED 专用样品制备 – Eisenberg（主导） 概括 微晶电子衍射 (MicroED) 的样品制备具有挑战性，并且是一个限速步骤。 在这里，我们将开发生长纳米晶体的方法，优化它们，并制定一个协议 MicroED 的可重复网格制备 目前，我们使用单颗粒制备 MicroED 样品。 冷冻电镜实验方案包括将样品溶液移至电镜网格上，吸干多余的溶液并冷冻。 通过将网格浸入液体乙烷中来对样品进行处理，此过程对样品的影响很大，尤其是吸墨。 可以将其暴露于水-空气界面，从而损害其结构并破坏下面的晶格。 对于膜蛋白来说，蛋白质“软”晶体的生长加剧了这个问题。 此外，在脂质立方相中生长晶体更具挑战性，因为它们嵌入。 进入粘稠的脂质基质中，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 在此，我们提出了由选定的生物项目挑战和测试的联合和系统的努力。 系统地测试纳米晶体生长的条件并确定哪些程序可以产生纳米晶体 我们将制定纳米晶体低温保护的新策略并开发纳米晶体生长。 最后，我们将通过一系列实验来决定最佳实践是什么。 FIB 研磨服务 MicroED 作为制备在脂质立方体中生长的膜蛋白纳米晶体的策略 我们将通过三个目标来实现这些目标：1.定向纳米晶体生长和检测；2. 3. 实验用 MicroED 网格准备策略 我们将为样品制备、检测和保存提供可重复且可靠的程序 MicroED 包括在 LCP 中生长的膜蛋白 长期目标是实现常规和高通量。 MicroED 的通量结晶和结构测定。