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

2017年诺贝尔奖认可的单粒子低温电子显微镜(Cryo-EM) 在化学方面，我们研究小组的Joachim Frank博士使用了电子显微镜(EM)对 通过快速低温冷却嵌入玻璃冰中的生物分子。时间分辨低温电子显微镜(TRCEM)， 低温电子显微镜的一种扩展，在这种扩展中，通过一系列的反应来检查样品 受控持续时间，允许研究生物分子事件的短暂中间状态，并具有 最近成功地应用于研究几种具有反应时间的细菌翻译系统 范围在20到600毫秒之间。然而，在更高时间分辨率下进行TRCEM研究是不可能的 由于现有的样品制备方法无法实现低于20ms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牵涉到 激活和选通时间在1毫秒或更短的人类疾病。此外，现有的低温EM 样品制备方法使用的设备都是硬连接的，用于特定的反应时间，即 效率低下，并可能导致样本质量不一致。 我们将开发一种微流控冷冻-EM样品制备系统，称为高均匀度和 解析反应中间体(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阵与均匀的反应控制和喷雾相结合 冷冻(简称反应-冷冻)装置。微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电磁网格上。在……里面 在反应冷冻装置中，栅格迅速转移到附近的制冷剂喷雾中，在到达反应物的途中 分子的反应时间很短，与它们在网格上的位置无关，直到被玻璃化。结合了 微喷雾器和反应冷冻技术将整个反应区的长度降至最低 不同的反应物分子所经历的反应时间的变化。因此，赫里将有能力 以高分辨率和低分散度生成亚毫秒级反应时间的样品。此外， 反应时间将是可调的，因为给定的Hurri乐器将在不需要修改任何 物理成分，允许在研究所需的一系列反应时间内产生样品。这个 Hurri操作的反应物流速也将比#年使用的 现有的标本制备方法，从而减少了生物材料的使用。因此，胡里 技术将以前所未有的速度实现对快速反应生物系统的高效TRCEM研究 高时间分辨率。Hurri的这种潜在的变革性效用将通过TRCEM案例进行演示 1型Ryanodine受体/钙释放通道(RyR1)是已知的最大的离子通道，是 是许多关键细胞功能所必需的。

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.