近原子分辨率冷冻电镜单颗粒重构方法研究与应用

发布时间：2018-03-11 11:01

本文选题：冷冻电子显微学　切入点：单颗粒重构技术　出处：《清华大学》2016年博士论文　论文类型：学位论文

【摘要】：结构生物学作为现代生命科学的前沿和带头学科之一,在对生命现象与规律的研究中发挥着举足轻重的作用。结构生物学研究手段主要有X-射线晶体学,核磁共振波谱学(NMR)及冷冻电子显微学(Cryo-EM)等,每种方式都有其相应的特点。X-射线晶体学适用于分子量较小的蛋白质,需要蛋白质能够形成规则的三维晶体,利用晶体衍射数据解析得到三维结构。核磁共振波谱学可以测定不同溶液状态下的结构,用于研究分子的柔性、分子间的相互作用、分子的动态特征等,但是对于分子量的局限性很大,只适用于分子量比较小的蛋白质(40-50 kDa)。相比之下,冷冻电镜技术不需要蛋白质结晶,且适用于分子量较大的蛋白质,因此成为研究生物大分子结构的首选。由于冷冻电镜成像技术及数据处理方式的发展,直接电子探测器、图像漂移校正算法及三维分类技术的出现,使得该领域取得了突破性进展。近两年涌现出了大批冷冻电镜高分辨结构,冷冻电镜逐渐成为结构生物学中最重要的研究手段之一。尽管冷冻电镜解析高分辨结构已经成为可能,但是对于如何进行结构解析,如何优化解析过程仍有许多问题需要解决。针对不同的研究对象,具体的结构解析方案也不尽相同,需要经过摸索和尝试才能找到最合适的方法解析得到高分辨结构。本论文建立和完善了有关高分辨冷冻电镜单颗粒的重构方法,尤其是螺旋体单颗粒重构方法。论文中首先对电子显微学成像原理及三维重构原理进行简单介绍。在此基础之上,以人源T4-γ-secretase及人源Rad51与DNA形成的螺旋纤维结构解析为例,详细阐述冷冻电子显微学高分辨结构解析如何进行。在结构解析过程中,进行了多种尝试,最终获得近原子分辨率结构,通过原子模型搭建及结构分析,为其生物学功能提供了结构基础,同时也为相应的生物学问题提供了结构依据。在人源T4-γ-secretase中解析得到了4.32?的结构,电镜结构揭示了γ-secretase四组分的组装机制,为后续γ-secretase的结构及功能研究提供了结构依据。在Rad51课题中,解析得到了pre-synaptic和post-synaptic复合物高分辨结构,获得了链间交换的可能中间态结构,为Rad51介导的DNA修复提供了结构基础。上述例子中,高分辨结构解析方法是根据样品摸索得到的,说明冷冻电镜高分辨结构解析仍需要进行方法学探索,冷冻电镜单颗粒重构方法研究具有重要意义。
[Abstract]:As one of the leading subjects in modern life sciences, structural biology plays an important role in the study of life phenomena and laws. Nuclear Magnetic Resonance Spectroscopy (NMR) and Frozen Electron Microscopy (Cryo-EMN), each method has its own characteristics. X- ray crystallography is suitable for proteins with small molecular weights, and requires proteins to form regular three-dimensional crystals. The three-dimensional structure can be obtained by using crystal diffraction data. Nuclear magnetic resonance spectroscopy can be used to study the flexibility of molecules, the interaction between molecules, the dynamic characteristics of molecules, and so on. However, the limitation of molecular weight is very great, and it is only suitable for proteins with relatively small molecular weight, which is 40-50 kDa. By contrast, the cryopreservation electron microscopy does not require protein crystallization, and is suitable for proteins with high molecular weight. As a result of the development of cryo-electron microscopy imaging technology and data processing, direct electronic detectors, image drift correction algorithms and three-dimensional classification techniques have emerged. This has made a breakthrough in this field. In the last two years, a large number of high resolution structures have emerged. Cryopreservation electron microscopy has gradually become one of the most important research methods in structural biology. There are still many problems to be solved on how to optimize the parsing process. It is necessary to find the most suitable method to analyze and obtain the high resolution structure. In this paper, the reconstruction method of single particle in high resolution cryopreservation electron microscope is established and improved. In this paper, the principle of electron microscopy imaging and the principle of three-dimensional reconstruction are briefly introduced. On this basis, the structure analysis of helical fibers formed by human T4- 纬 -secretase and human Rad51 and DNA is taken as an example. In the process of structure resolution, many attempts were made to obtain the near atomic resolution structure, and the atomic model was built and the structure was analyzed. It provides the structural basis for its biological function, and also provides the structural basis for the corresponding biological problems. In human T4- 纬 -secretase, 4.32? The structure and electron microscope structure of 纬 -secretase revealed the assembly mechanism of 纬 -secretase, which provided the structural basis for further study on the structure and function of 纬 -secretase. In the Rad51 project, the high-resolution structures of pre-synaptic and post-synaptic complexes were obtained. The possible intermediate structure of interchain exchange is obtained, which provides a structural basis for DNA repair mediated by Rad51. It shows that the resolution of high resolution structure of cryopreservation electron microscope still needs to be explored, and it is of great significance to study the method of single particle reconstruction of cryopreservation electron microscope.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q617

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