Norville Julie E, Kelly Deborah F, Knight Thomas F, Belcher Angela M, Walz Thomas
MIT Computer Science and Artificial Intelligence Laboratory, 32 Vassar Street, Cambridge, MA 02139, USA.
J Struct Biol. 2007 Dec;160(3):313-23. doi: 10.1016/j.jsb.2007.06.002. Epub 2007 Jun 15.
Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.
脂质单分子层上的二维结晶是通过电子显微镜获取蛋白质结构信息的通用工具。这种方法的一个固有问题是，要以一种既能保持蛋白质阵列的晶体秩序，又能制备出足够平整、以便在高倾斜角度下进行高分辨率数据采集的标本的方式来制备样品。作为优化用于电子显微镜成像的脂质单分子层晶体制备的测试标本，我们使用了S层蛋白sbpA，该蛋白有潜力设计具有工程特性的生物和无机材料阵列，用于各种纳米技术应用。目前认为糖包埋是制备重构到脂质双层中的膜蛋白二维晶体的最佳方法。我们发现，使用环将脂质单分子层晶体转移到电子显微镜网格上，然后用海藻糖包埋并在液态乙烷中快速冷冻，也能为sbpA脂质单分子层晶体产生最高分辨率的图像。利用以这种方式制备的标本图像，我们可以计算出分辨率为7埃的sbpA投影图，这是迄今为止用脂质单分子层晶体获得的最高分辨率投影结构之一。
