用于电子晶体学的整合膜蛋白二维结晶
Two-dimensional crystallization of integral membrane proteins for electron crystallography.
作者信息
Stokes David L, Rice William J, Hu Minghui, Kim Changki, Ubarretxena-Belandia Iban
机构信息
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA.
出版信息
Methods Mol Biol. 2010;654:187-205. doi: 10.1007/978-1-60761-762-4_10.
Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microscopy.
尽管膜蛋白占蛋白质组的30%，并且是治疗药物的常见靶点，但其原子结构的确定仍然是一项技术挑战。电子晶体学是传统X射线衍射和核磁共振方法之外的另一种方法，它依赖于二维晶体的形成。这些晶体是通过将纯化的、去污剂溶解的膜蛋白重新构建到脂质双层的天然环境中产生的。本章综述了二维晶体的制备方法以及通过负染电子显微镜对其进行筛选的方法。此外，我们展示了常见获得的不同形态的实例，并描述了可用于评估其通过冷冻电子显微镜进行结构测定前景的基本图像分析程序。
Zotero 插件