Technology for sample preparation and storage for cryo-electron microscopy

项目摘要 由于过去十年的技术进步，单粒子冷冻电子显微镜(Cryo-EM)已经 作为获得大型生物分子络合物的近原子分辨结构的一种有效方法， 膜蛋白，以及其他生物和生物医学上重要的靶点，在许多情况下 对X射线结晶学来说很难处理。冷冻-EM只需要少量分散在溶液中的生物分子 并且已经成为结构确定的初始尝试的首选。巨大的投资一直是 并继续在大学和生物医学研究的新显微镜和低温电磁设备中制造 在美国和世界各地的研究所。然而，目前的样品制备和处理方案-主要是 基于20世纪80年代以来的工作，这些方法复杂、耗时、容易出错，而且往往产生不太理想的结果。 它们是限制这些昂贵的新设施有效利用的主要因素。 这个第一阶段的SBIR项目建立在对冷冻、玻璃化和 多组分系统的热机械响应，以及MiTeGen在开发方面的丰富专业知识 用于微量样品处理和冷冻冷却的工具和仪器。新的样品保持支架将是 开发了提高最大样品冷却速度和样品质量，降低机械应力在 冷却是电子束中降低分辨率的样品运动的主要来源，有助于 样品制备和冷冻过程的表征和优化，并使样品 身份识别和跟踪。将开发一种新的自动样品冷却仪器，使用液体 氮气而不是液体乙烷作为主要的样品冷却剂，同时提供出色的样品玻璃化 性能。该项目的长期目标是提供一个集成的冷冻-EM解决方案，包括 改进了用于安全处理、存储和跟踪的样品支架和工具，改进了样品工具 稀化/去除，以及具有集成样品跟踪的自动样品冷冻和冷冻存储系统 这消除了人工操作，提供了稳定、无污染的冷链，从样品下落到 这大大提高了低温EM结构测定管道的效率。

Project Summary As a result of technical advances in the last decade, single-particle cryo-electron microscopy (cryo-EM) has emerged as a powerful approach to obtaining near atomic resolution structures of large biomolecular complexes, membrane proteins, and other biologically and biomedically important targets that in many cases have been intractable to X-ray crystallography. Cryo-EM requires only small amounts of biomolecule dispersed in solution and has become a first choice for initial attempts at structure determination. Enormous investments have been and continue to be made in new microscopes and cryo-EM facilities at universities and biomedical research institutes in the US and around the world. However, current sample preparation and handling protocols – largely based on work from the 1980s, are complex, time consuming, error-prone, and often yield suboptimal outcomes. They are a major factor limiting efficient use of these expensive new facilities. This Phase I SBIR project builds on insights into fundamental processes of cryocooling, vitrification, and the thermomechanical response of multicomponent systems, and on MiTeGen's extensive expertise in development of tools and instruments for microsample manipulation and cryocooling. New sample holding supports will be developed that increase maximum sample cooling rates and sample quality, reduce mechanical stresses during cooling that are the primary source of resolution-degrading sample motion in the electron beam, facilitate characterization and optimization of sample preparation and cryocooling processes, and enable sample identification and tracking. A new automated sample cooling instrument will be developed that uses liquid nitrogen rather than liquid ethane as the primary sample coolant while delivering outstanding sample vitrification performance. The longer-term goal of this project is to deliver an integrated cryo-EM solution comprised of improved sample supports and tools for their safe handling, storage, and tracking, improved tools for sample thinning/removal, and an automated sample cryocooling and cryostorage system with integrated sample tracking that eliminates manual manipulations, provides a stable, contaminant-free cold chain from sample plunging to the microscope, and that substantially improves the efficiency of cryo-EM structure determination pipelines.