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°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 ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.

中文翻译：

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在室温和低温条件下对蛋白质晶体进行高通量原位X射线筛选并从中收集数据。

近年来，蛋白质晶体学取得了显着进步，其中就地数据收集是重点关注的问题，在这些数据收集中，晶体被放置在生长介质中的X射线束中。原位方法消除了收获晶体的需要，这是以前无法避免的缺点，特别是对于通常较小的膜蛋白晶体而言。在这里，我们提出了一种用于在室温（20-25°C）和低温条件下从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协议。Mylar原位法使用基于Mylar的胶片夹层板，该板价格便宜，易于制造且与自动成像兼容，并且背景散射极低。它们支持微批量和气相扩散模式以及脂质立方相（LCP）的结晶。一套用于不同尺寸的Mylar三明治薄膜补丁的3D打印支架使该方法既坚固又通用，可以存储和运输晶体，并可以自动安装在同步加速器上，以及基于测角计的筛选和数据收集。该方案涵盖原位板的制备和结晶试验的设置；支架的3D打印和组装; 打开印版，隔离包含晶体的胶片，并将其装载到支架上；基本筛选和数据收集指南；持有人的卸货，以及对他们的再利用和回收。原位板在1小时内准备好并组装；支架经过3D打印并在≤90分钟内组装完毕；并打开原位平板，分离出包含晶体的薄膜贴片，并在5分钟内将其加载到支架上。