Microfluidic Preparation of Specimens to Enable Submillisecond Time-Resolved Cryo-EM

样品的微流体制备以实现亚毫秒时间分辨冷冻电镜

• 批准号: 10736937
• 负责人: Qiao Lin
• 金额: $ 49.26万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736937
• 关键词: Address; Award; Bacteria; Binding; Biocompatible Materials; Biological; Case Study; Cell physiology; Chemistry; Consumption; Cryoelectron Microscopy; Deposition; Devices; Disease; Electron Microscopy; Eukaryota; Event; Gases; Generations; Ice; Individual; Ion Channel; Length; Methods; Microfluidics; Modification; Nobel Prize; Positioning Attribute; Preparation; Reaction; Reaction Time; Research; Research Design; Resolution; Route; RyR1; Ryanodine Receptor Calcium Release Channel; Sampling; Series; Signal Transduction; Site; Specimen; Speed; System; Techniques; Technology; Therapeutic; Time; Translations; Travel; Uncertainty; Variant; Visualization; biological systems; cryogenics; empowerment; experience; human disease; instrument; millisecond; particle; receptor; residence; structural biology; temporal measurement; tool

Single-particle cryogenic electron microscopy (cryo-EM), recognized by the award of the 2017 Nobel Prize in Chemistry to Dr. Joachim Frank of our research team, uses electron microscopy (EM) of specimens of biological molecules embedded in vitreous ice via rapid cryogenic cooling. Time-resolved cryo-EM (TRCEM), an expansion of cryo-EM in which specimens are examined resulting from reactions occurring for a series of controlled time durations, allows studies of short-lived intermediate states of biomolecular events and has recently been applied successfully to investigate several bacterial translation systems with reaction times ranging between 20 and 600 ms. TRCEM studies at higher time resolutions, however, have not been possible as existing specimen preparation methods preclude reaction times below 20 ms from being achieved. This capability gap is highly limiting, because there are a large number of ion channels and receptors implicated in human diseases with activation and gating times on the order of 1 ms or less. In addition, existing cryo-EM specimen preparation methods use devices that are each hardwired for a particular reaction time, which is inefficient and may cause inconsistencies in specimen quality. We will develop a microfluidic cryo-EM specimen preparation system, termed the High Uniformity and Resolution Reaction Intermediates (HURRI) system, to address these challenges. The system consists of a microsprayer or a line array of microsprayers in combination with a uniform reaction control and spray cryocooling (in short, react-cryocool) unit. The microsprayer rapidly mixes two reactants at a low flow rate, atomizes the mixture into monodisperse droplets, and delivers and deposits the droplets onto an EM grid. In the react-cryocool unit, the grid is rapidly transferred into a nearby cryogen spray, en route to which reactant molecules react for a short time independent of their on-grid position until being vitrified. Combining the microsprayer and react-cryocool technologies minimizes the length of the overall reaction zone as well as variations in the reaction time experienced by different reactant molecules. Thus, HURRI will be capable of generating specimens with submillisecond reaction times at high resolution and low dispersity. In addition, the reaction time will be tunable in that a given HURRI instrument will, without requiring modifications of any physical components, allow generation of specimens at a series of reaction times needed for a study. The reactant flow rates at which HURRI operates will also be one order of magnitude lower than those used in existing specimen preparation methods, thereby reducing the use of biological material. As such, the HURRI technology will enable highly efficient TRCEM studies of fast-reacting biological systems at an unprecedented high time resolution. This potentially transformative utility of HURRI will be demonstrated with a TRCEM case study of type 1 ryanodine receptor/calcium release channels (RyR1), the largest known ion channel that is required for numerous critical cellular functions.

单粒子低温电子显微镜（cryo-EM）获得2017年诺贝尔奖 在化学博士约阿希姆弗兰克我们的研究小组，使用电子显微镜（EM）的标本， 通过快速低温冷却将生物分子嵌入玻璃冰中。时间分辨冷冻电镜（TRCEM）， 冷冻电镜的一种扩展，其中检查标本是由一系列 受控的持续时间，允许研究生物分子事件的短暂中间状态， 最近成功地应用于研究几种细菌翻译系统的反应时间 在20和600毫秒之间，然而，在更高的时间分辨率下的TRCEM研究是不可能的 因为现有的样品制备方法阻止了实现低于20毫秒的反应时间。这 能力差距是非常有限的，因为有大量的离子通道和受体涉及 激活和门控时间大约为1 ms或更少的人类疾病。此外，现有的冷冻EM 样品制备方法使用的装置都是硬连线的，用于特定的反应时间， 效率低，并可能导致样品质量的不一致。 我们将开发一个微流控冷冻电镜标本制备系统，称为高均匀性， 拆分反应中间体（HURRI）系统，以应对这些挑战。该系统由一个 微喷雾器或微喷雾器的线性阵列与均匀反应控制和喷雾组合 低温冷却（简称反应-低温冷却）装置。微型喷雾器以低流速快速混合两种反应物， 将混合物雾化成单分散液滴，并将液滴输送和沉积到EM网格上。在 在反应-低温冷却单元中，栅格被快速地转移到附近的冷冻剂喷雾中，在到达该反应物的途中， 分子反应很短的时间，与它们在栅格上的位置无关，直到玻璃化。结合 微型喷雾器和反应低温冷却技术最大限度地减少了整个反应区的长度， 不同反应物分子经历的反应时间的变化。因此，HURRI将能够 以高分辨率和低分散性产生具有亚毫秒反应时间的样品。此外该 反应时间将是可调的，因为给定的HURRI仪器将不需要任何修改， 物理组件，允许在研究所需的一系列反应时间生成样本。的 HURRI操作的反应物流速也将比在HURRI中使用的反应物流速低一个数量级。 现有的标本制备方法，从而减少生物材料的使用。因此，人权委员会 技术将使快速反应生物系统的高效TRCEM研究以前所未有的速度进行。 高时间分辨率。这种潜在的变革效用的HURRI将证明与TRCEM的情况下 1型兰尼碱受体/钙释放通道（RyR 1）的研究，RyR 1是已知的最大的离子通道， 许多重要的细胞功能所必需的。