A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals.
A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals.
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DOI：
10.1021/acs.cgd.6b00950
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发表时间：
2016-11-02
影响因子：
3.8
通讯作者：
Ernst OP
中科院分区：
文献类型：
--
作者：
Broecker J;Klingel V;Ou WL;Balo AR;Kissick DJ;Ogata CM;Kuo A;Ernst OP
近年来，原位数据收集一直是蛋白质晶体学进展的一个主要焦点。在这里，我们介绍了聚酯薄膜原位方法，使用聚酯薄膜为基础的夹层板，价格低廉，易于制造和处理，并显示显着减少背景散射比其他设置。用于对应于一个或多个威尔斯孔的Mylar原位夹心膜贴片的各种同源保持器使得该方法稳健且通用，允许整个威尔斯孔的储存和运输，并且能够在室温和低温条件下对在这些板中生长或转移到这些板的可溶性和膜蛋白晶体进行自动晶体成像、筛选和基于测角计的X射线衍射数据收集。我们验证了聚酯薄膜原位方法使用的晶体的水溶性蛋白质鸡蛋白溶菌酶和抹香鲸肌红蛋白，以及7-跨膜蛋白细菌视紫红质Haloquadratum walsbyi。结合目前同步加速器的发展，这种方法有望使膜蛋白的高分辨率结构研究变得更快，更常规。我们介绍了聚酯薄膜原位方法，使用聚酯薄膜为基础的夹层板，这是优于既定的设计。同源支架使该方法稳健且通用，并允许在室温和低温条件下对在这些板中生长或转移到这些板上的可溶性和膜蛋白晶体进行自动晶体成像、筛选和基于测角计的X射线衍射数据收集。
In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine. We introduce the Mylar in situ method that uses Mylar-based sandwich plates, which are advantageous over established designs. Cognate holders make the method robust and versatile and allow for automated crystal imaging, screening, and goniometer-based X-ray diffraction data collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates.