Gentle, fast and effective crystal soaking by acoustic dispensing
Gentle, fast and effective crystal soaking by acoustic dispensing
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DOI：
10.1107/s205979831700331x
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发表时间：
2017-03-01
影响因子：
2.2
通讯作者：
von Delft, Frank
中科院分区：
文献类型：
--
作者：
Collins, Patrick M.;Ng, Jia Tsing;von Delft, Frank
通过提高X射线亮度、机器人能力和探测器速度，现代光束线用于高通量数据收集的能力稳步扩大，将瓶颈推向了样品制备的上游。即使在使用晶体浸泡的配体结合研究中，该实验最能利用光束线能力，主要限制是需要温和和非平凡的浸泡方案，例如逐步增加浓度，即使对于稳健和良好表征的晶体。在此，描述了使用声学液滴喷射来用小分子浸泡蛋白质晶体，并且表明其对晶体温和并且允许非常高的生产量，在10分钟内容易地进行1000次独特的浸泡。（每个样品数十纳升），声学液滴喷射的位置精度使得能够将化合物/溶剂远离晶体并朝向液滴边缘定向放置，允许溶剂逐渐扩散通过液滴。这确保了X射线衍射的再现性的改善和晶体的溶剂耐受性的增加，从而实现更高的有效化合物浸泡浓度。该技术在这里详细介绍了来自蛋白质靶点JMJD 2D的例子，JMJD 2D是一种在癌症中发挥作用的组蛋白赖氨酸脱甲基酶，也是基于活性结构的药物设计工作的重点。
The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compoundsoaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.