2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays.
2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays.
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膜蛋白单层、双层和多层有序阵列的二维电子晶体学。
DOI：
10.1007/978-1-0716-0966-8_10
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Schmidt-Krey,Ingeborg
中科院分区：
文献类型：
--
作者：
Johnson,MatthewC;Uddin,YusufM;Neselu,Kasahun;Schmidt-Krey,Ingeborg
二维电子晶体学的电子冷冻显微镜（cryo-EM）方法允许可溶性和膜蛋白的二维（2D）晶体的结构测定，一旦获得2D晶体，则采用相同的原理和方法。膜蛋白的二维结晶试验可以产生有序阵列的多种结果，其可以适合于二维电子晶体学、螺旋分析或MicroED.The膜蛋白2D晶体用于2D电子晶体学是单层或双层有序的蛋白脂质体囊泡或片状膜。我们研制出了一种低温电镜 网格制备方法，允许分析既不适合MicroED的堆叠2D晶体 也不用于直接应用2D电子晶体学。这种新的网格制备方法，即剥离印迹法，使用亚微米滤纸产生的毛细管力和机械手段将堆叠的2D晶体分离成单层2D晶体，然后可以采用标准的2D电子晶体学。本文介绍了二维晶体的制备、剥离网格的制备和二维电子晶体学的结构测定。
The electron cryo-microscopy (cryo-EM) approach of 2D electron crystallography allows for structure determination of two-dimensional (2D) crystals of soluble and membrane proteins, employing identical principles and methods once 2D crystals are obtained. Two-dimensional crystallization trials of membrane proteins can result in multiple outcomes of ordered arrays, which may be suited for either 2D electron crystallography, helical analysis, or MicroED.The membrane protein 2D crystals used for 2D electron crystallography are either single- or double-layered ordered proteoliposome vesicles or sheet-like membranes. We have developed a cryo-EM grid preparation approach, which allows for the analysis of stacked 2D crystals that are neither suitable for MicroED nor for directly applying 2D electron crystallography. This new grid preparation approach, the peel-blot, uses the capillary force generated by submicron filter paper and mechanical means for the separation of stacked 2D crystals into single-layered 2D crystals, for which standard 2D electron crystallography can then be employed. The preparation of 2D crystals, the peel-blot grid preparation, and the structure determination by 2D electron crystallography are described here.