High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions
High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions
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DOI：
10.1038/nprot.2017.135
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发表时间：
2018-02-01
期刊：
影响因子：
14.8
通讯作者：
Ernst, Oliver P.
中科院分区：
文献类型：
--
作者：
Broecker, Jana;Morizumi, Takefumi;Ernst, Oliver P.
蛋白质晶体学近年来取得了显著进展，原位数据收集是一个主要的焦点，其中晶体被放置在生长介质中的x射线束中。原位法消除了收集晶体的需要，这是以前不可避免的缺点，特别是对于通常较小的膜蛋白晶体。在这里，我们提出了一种在室温（20-25℃）和低温条件下对可溶性和膜蛋白晶体进行高通量原位x射线筛选和数据收集的方案。Mylar原位法使用基于Mylar的薄膜夹层板，价格低廉，易于制作，与自动成像兼容，并且显示出非常低的背景散射。它们支持微批和蒸汽扩散模式以及脂质立方相（lcp）的结晶。一组3d打印支架用于不同尺寸的聚酯薄膜夹层膜片，使该方法坚固耐用，用途广泛，允许晶体的存储和运输，并能够自动安装在同步加速器上，以及基于角计的筛选和数据收集。该方案包括原位板的制备和结晶试验的设置；支架3D打印组装；打开印版，隔离含有晶体的薄膜片，并将其装入支架上；基本筛选和数据收集准则；和卸载支架，以及它们的再利用和回收。现场板在1小时内制备和组装；支架是3d打印和组装在
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25 degrees C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in