Han Bong-Gyoon, Watson Zoe, Kang Hannah, Pulk Arto, Downing Kenneth H, Cate Jamie, Glaeser Robert M
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
J Struct Biol. 2016 Aug;195(2):238-244. doi: 10.1016/j.jsb.2016.06.009. Epub 2016 Jun 15.
We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.
我们描述了一种直接在有孔碳电子显微镜网格上生长链霉亲和素（SA）单层晶体的快速便捷方法。正如预期的那样，这些SA单层晶体通过在海藻糖中包埋以及随后去除海藻糖的循环过程，保留了它们的生物素结合功能和晶体有序性。这一事实使得人们能够制备并储存电子显微镜网格，其中SA单层晶体作为用于制备生物大分子标本的亲和底物以供后续使用。此外，我们报告称，用蒸发碳涂覆海藻糖包埋的单层晶体的脂尾侧似乎能提高观察到排列良好的单晶跨越支撑膜整个2μm孔洞的一致性。使用随机生物素化的70S核糖体作为测试标本，以表明这些支撑膜可用于获得高分辨率的冷冻电子显微镜结构。
