Technology for sample preparation and storage for cryo-electron microscopy

冷冻电子显微镜样品制备和储存技术

• 批准号: 10010925
• 负责人: Benjamin Arthur Apker
• 金额: $ 22.5万
• 依托单位: MITEGEN, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10010925
• 关键词: Bar Codes; Biological; Biomedical Research; Cold Chains; Complex; Consumption; Cryoelectron Microscopy; Development; Diagnosis; Electron Beam; Ethane; Excision; Film; Goals; Ice; Individual; Investments; Liquid substance; Manuals; Measurement; Mechanical Stress; Mechanics; Membrane Proteins; Methods; Microscope; Motion; Nitrogen; Outcome; Performance; Phase; Preparation; Process; Protocols documentation; Reproducibility; Research Institute; Resolution; Sampling; Small Business Innovation Research Grant; Source; Structure; Surface; System; Technology; Testing; Thick; Thinness; Time; United States National Institutes of Health; Universities; Work; X-Ray Crystallography; base; crystallinity; design; improved; innovation; insight; instrument; interest; particle; prototype; response; support tools; tool; tool development

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.

项目摘要 作为过去十年技术进步的结果，单粒子冷冻电子显微镜（冷冻EM） 作为一种获得大生物分子复合物的近原子分辨率结构的强有力的方法出现， 膜蛋白和其他生物学和生物医学上重要的目标，在许多情况下， X射线晶体学无法解释的Cryo-EM只需要少量的生物分子分散在溶液中 并且已经成为结构测定的初始尝试的首选。巨额投资已经 并继续在大学和生物医学研究的新显微镜和冷冻EM设施中制造 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-在很大程度上 基于20世纪80年代的工作，是复杂的，耗时的，容易出错的，而且往往产生次优的结果。 它们是限制有效利用这些昂贵的新设施的一个主要因素。 这个第一阶段SBIR项目建立在对低温冷却，玻璃化和冷冻的基本过程的深入了解之上。 多组分系统的热机械响应，以及MiTeGen在开发方面的广泛专业知识 用于显微样品操作和低温冷却的工具和仪器。新的样品固定支架将 提高最大样品冷却速率和样品质量，降低 冷却是电子束中分辨率降低的样品运动的主要来源， 表征和优化样品制备和低温冷却过程，并使样品 识别和跟踪。将开发一种新的自动样品冷却仪器， 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 改进的样品支架和工具，用于安全处理、储存和跟踪，改进的样品工具 减薄/去除，以及具有集成样品跟踪的自动样品低温冷却和低温储存系统 无需人工操作，提供稳定、无污染的冷链，从样品投入到 显微镜，并大大提高了效率的冷冻EM结构确定管道。