Transmission Electron Microscopy of Crystallization of Lysozyme in a Solution
Transmission Electron Microscopy of Crystallization of Lysozyme in a Solution
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溶液中溶菌酶结晶的透射电子显微镜
DOI：
10.1017/s143192761500207x
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发表时间：
2015
影响因子：
2.8
通讯作者：
H. Matsumoto and Y. Kimura
中科院分区：
文献类型：
--
作者：
T. Yamazaki;M. Shirai;H. Matsumoto and Y. Kimura
作为材料科学的基础研究，观察蛋白质的结晶过程对于利用蛋白质晶体通过X射线衍射图谱分析蛋白质分子的结构具有重要意义。研究结晶过程的有用方法是原位显微镜观察，因为它可以直接可视化真实的过程。蛋白质结晶的观察主要使用光学显微镜进行[1, 2]，并且该过程的微观尺度已经得到很好的理解。然而，由于没有原位观察，纳米尺度的蛋白质结晶过程仍不清楚。近年来，人们积极利用透射电子显微镜（TEM）结合液体池或离子液体对纳米粒子的行为和无机材料的结晶过程进行原位观察，并在纳米尺度上部分证明了这些机制[3-5]。然而，迄今为止还没有观察到蛋白质的结晶。我们使用TEM和液体池对蛋白质晶体进行了原位观察，以了解其结晶过程。我们使用鸡蛋清溶菌酶作为蛋白质样品。使用NaCl作为沉淀剂在pH＝4.5的乙酸钠缓冲溶液中使溶菌酶结晶。为了进行观察，我们使用了带有两个输入端口和一个输出端口的“Poseidon”液体池支架（Protochips Inc.）。液体池由一对带有非晶氮化硅窗口的半导体板和 150 或 500 nm 厚的间隔物组成，以形成结晶溶液的流动路径。我们使用了两台带有 LaB6 灯丝的 TEM，加速电压为 200 kV (Hitachi H-8100)，并配有场发射枪，加速电压为 300 kV (Hitachi HF-3300)。
It is important to observe the crystallization process of proteins as the fundamental study of the material science and for the structure analysis of a protein molecule by X-ray diffraction pattern using a protein crystal. The useful method to study the crystallization process is in-situ microscope observation because it can directly visualize the real process. The observation of protein crystallization have been performed mainly using optical microscopy [1, 2] and the micro-scale view of the process have been well understood. However, the protein crystallization process at nano-scale is still unclear because there is no in-situ observation of it. Recently, in-situ observations of the behavior of nano-particles and the crystallization processes of inorganic materials have been energetically performed by the transmission electron microscopy (TEM) combined with the liquid cell or an ionic liquid, and these mechanisms at nano-scale are partly demonstrated [3-5]. However, there are no observations of crystallization of proteins so far. We performed in-situ observation of the protein crystal for understanding its crystallization process using TEM with the liquid cell.We used the hen-egg white lysozyme as a protein sample. The lysozyme was crystallized using NaCl as a precipitant in a sodium acetate buffer solution at pH= 4.5. For the observation, we used a “Poseidon” liquid cell holder (Protochips Inc.) with two input and one output ports. The liquid cell consists of a pair of semiconductor-based plates with an amorphous silicon nitride window and 150 or 500-nm-thick spacer to form the flow path of the crystallization solution. We used two TEMs with LaB6 filament at an acceleration voltage of 200 kV (Hitachi H-8100) and with field-emission gun at an acceleration voltage of 300 kV (Hitachi HF-3300).