High-throughput methods for electron crystallography.
High-throughput methods for electron crystallography.
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电子晶体学的高通量方法。
DOI：
10.1007/978-1-62703-176-9_15
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Engel,Andreas
中科院分区：
文献类型：
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作者：
Stokes,DavidL;Ubarretxena-Belandia,Iban;Gonen,Tamir;Engel,Andreas
膜蛋白在细胞生理学中发挥着极其重要的作用，并成为越来越多药物的靶点。结构信息是了解它们的功能和制定抗击疾病的新战略的关键。然而，与膜蛋白相关的复杂物理化学使它们比它们的可溶性近亲更难研究。电子结晶学历来是解决膜蛋白质结构的一种成功方法，其优点是为这些蛋白质提供了一个天然的脂环境。具体地说，当膜蛋白在脂质双层内形成二维阵列时，可以使用电子显微镜来收集图像和衍射，并结合相应的数据来产生三维重建，在有利的条件下，这种重建可以扩展到原子分辨率。像X射线结晶学一样，结构的质量在很大程度上取决于晶体的顺序和大小。然而，与X射线结晶学不同的是，用于筛选电子结晶学结晶试验的高通量方法并不普遍使用。在本章中，我们描述了两种高通量筛选脂双层中膜蛋白结晶的方法。第一种方法依靠传统的透析法去除洗涤剂，从而重建双层；标准96孔格式的透析孔阵列允许使用液体处理机器人，并极大地提高了吞吐量。第二种方法依靠滴定环糊精作为洗涤剂的螯合剂；设计了一种专门的移液机器人，不仅可以系统地添加环糊精，而且可以用光散射来监测重建过程。此外，还描述了使用液体处理机器人制作负色栅格以及在电子显微镜中自动成像样品的方法。
Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.