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 - Eisenberg （Lead）专用样品制备