TRD1: Dedicated sample preparation for MicroED

TRD1：MicroED 专用样品制备

• 批准号: 10641815
• 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/10641815
• 关键词: Adopted; Air; Alcohols; Biological; Biological Assay; Cryoelectron Microscopy; Cryopreservation; Crystallization; Dedications; 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 的可重复网格准备。目前，我们使用单粒子制备 MicroED 样品 冷冻电镜协议。这包括将样品溶液移至 EM 网格上，吸干多余的溶液，然后冷冻 将网格浸入液体乙烷中以获取样品。这个过程对样品非常苛刻，尤其是印迹 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。 脂质或清洁剂。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 受到选定的生物项目的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可以产生纳米晶体 最高品质。我们将建立纳米晶低温保护新策略并开发纳米晶技术 和保存套件。最后，我们将通过一系列实验来决定什么是最佳实践 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 阶段。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. 纳米晶体低温保护策略； 3.实验用MicroED网格制备策略。全面的， 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 MicroED 的通量结晶和结构测定。