Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes
Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes
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DOI：
10.1107/s1399004713034603
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发表时间：
2014-05-01
影响因子：
2.2
通讯作者：
Soares, Alexei S.
中科院分区：
文献类型：
--
作者：
Yin, Xingyu;Scalia, Alexander;Soares, Alexei S.
声学液滴喷射（ADE）是一项强大的技术，支持晶体学应用，如生长，改善和操纵蛋白质晶体。描述了一种片段筛选策略，该策略使用ADE直接在MiTeGen MicroMeshes上与片段库共结晶蛋白质。共结晶试验可以快速、经济地制备。样品制备的高速度和片段和蛋白质的低消耗允许使用单个而不是汇集的片段。Echo 550液体处理仪器（Labcyte Inc., Sunnyvale, California， USA）产生具有精确轨迹的液滴，这使得多个共结晶实验可以离散地定位在单个数据收集微网格上。这种精度还允许所有组件通过小孔径转移。因此，在蛋白质、沉淀剂和碎片转移到将发生结晶的微孔之前、期间和之后，结晶盘与储液器处于平衡状态。这种对样品环境的严格控制意味着，当工作体积从几微升水平降低到几纳升水平时，晶体学实验保持不变。利用该系统，溶菌酶、热溶酶、胰蛋白酶和水胱甘肽去甲基酶晶体与一个小的33个化合物迷你文库共结晶，以搜索片段命中。这项技术推动了一种更快、更自动化、更灵活的基于结构的药物发现策略，每种主要成分只需2.5升。
Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.