Key Laboratory of Protein Sciences (Tsinghua University), Ministry of Education, Beijing, China; School of Life Sciences, Tsinghua University, Beijing, China.
Key Laboratory of Protein Sciences (Tsinghua University), Ministry of Education, Beijing, China; School of Life Sciences, Tsinghua University, Beijing, China.
J Struct Biol. 2019 Mar 1;205(3):59-64. doi: 10.1016/j.jsb.2019.02.004. Epub 2019 Feb 20.
Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the electron crystallographic method on large protein crystals with micrometer or larger sizes. Here, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, including radiation damage on the crystal surface during the FIB imaging, deformation of the thin crystal lamella, and variation in the diffraction intensities under electron radiation. These observations provide important information to optimize the FIB milling, and hence is important to obtain high-quality crystal samples for routine structure determination of protein crystals using the electron cryo-microscope.
电子衍射为解决小蛋白晶体结构提供了有力工具。然而，电子与材料之间的强烈相互作用限制了电子晶体学方法在具有微米或更大尺寸的大蛋白晶体上的应用。在这里，我们使用配备在扫描电子显微镜（SEM）上的聚焦离子束（FIB）将大晶体铣成薄的薄片。观察和研究了铣削对晶体薄片的影响，包括 FIB 成像过程中晶体表面的辐射损伤、薄晶体薄片的变形以及电子辐射下衍射强度的变化。这些观察结果为优化 FIB 铣削提供了重要信息，因此对于使用电子冷冻显微镜常规确定蛋白质晶体结构获得高质量的晶体样品非常重要。
