School of Applied and Engineering Physics, Cornell University , Ithaca, New York 14853, USA.
Meinig School of Biomedical Engineering, Cornell University , Ithaca, New York 14853, USA.
Struct Dyn. 2016 Aug 29;3(5):054301. doi: 10.1063/1.4961971. eCollection 2016 Sep.
Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines.
蛋白质结构的知识为功能提供了重要的见解，增强了我们对疾病的理解，并为新的治疗方法的开发提供了可能。X 射线晶体学已被用于解析超过 100000 种蛋白质的结构；然而，绝大多数结构都代表了长寿状态，无法捕捉这些分子机器的功能运动。由于难以同步反应以产生可检测数量的瞬态状态，配体的加入引发的反应通常是最难用晶体学检测到的。X 射线自由电子激光（XFEL）和连续飞秒晶体学（SFX）的发展为解决蛋白质结构提供了新的方法，可在配体快速扩散到微米级蛋白质晶体后解析结构。毫秒级的构象变化可以被检测到并进行时间分辨。在这里，我们描述了一种新的 XFEL 注射器，它采用微流混合器，在样品到达 X 射线束之前几毫秒内快速混合反应物和样品。混合注射器由键合的同心玻璃毛细管组成。采用定制的激光切割中心间隔件和紫外光固化环氧树脂的制造工艺，确保了毛细管的精确对准，以实现可重复的、中心的样品流动和可靠的混合。晶体输送毛细管的直径为 50 或 75 μm，并可根据实验要求集成过滤器。反应时间可以从亚毫秒到几百毫秒不等。该注射器具有快速且均匀的混合、低样品稀释度和高命中率。它完全兼容现有的 SFX 光束线。
