Brink J, Gross H, Tittmann P, Sherman M B, Chiu W
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA.
J Microsc. 1998 Jul;191(Pt 1):67-73. doi: 10.1046/j.1365-2818.1998.00342.x.
Charging causes a loss of resolution in electron cryomicroscopy with biological specimens prepared without a continuous carbon support film. Thin conductive films were deposited onto catalase crystals prepared across holes using ion-beam sputtering and thermal evaporation and evaluated for the effectiveness of charge reduction. Deposits applied by ion-beam sputtering reduced charging but concurrently resulted in structural damage. Coatings applied by thermal evaporation also reduced charging, and preserved the specimen structure beyond 5 A resolution as judged from electron diffraction patterns and images of glucose-embedded catalase crystals tilted to 45 degrees in the microscope. This study demonstrates for the first time the feasibility of obtaining high-resolution data from unstained, unsupported protein crystals with a conductive surface coating.
对于未使用连续碳支撑膜制备的生物样本，在电子冷冻显微镜中充电会导致分辨率下降。使用离子束溅射和热蒸发将薄导电膜沉积到穿过孔洞制备的过氧化氢酶晶体上，并评估其减少电荷的效果。离子束溅射施加的沉积物减少了充电，但同时导致了结构损伤。热蒸发施加的涂层也减少了充电，并且根据电子衍射图和在显微镜中倾斜45度的葡萄糖包埋过氧化氢酶晶体的图像判断，其在超过5埃的分辨率下保留了样本结构。这项研究首次证明了通过导电表面涂层从未染色、无支撑的蛋白质晶体中获得高分辨率数据的可行性。
