基于同步辐射的串行晶体学实验技术研究

发布时间：2018-05-15 15:47

本文选题：串行晶体学 + 同步辐射　；参考：《中国科学院研究生院(上海应用物理研究所)》2017年硕士论文

【摘要】：同步辐射X射线衍射是当前蛋白质三维结构测定的最主要方法,可高效获取蛋白质晶体的高质量衍射数据。然而高亮度同步辐射对蛋白质晶体会产生辐射损伤,导致其结构信息减弱和改变。虽然晶体低温冷冻技术可以降低高强度的X射线光束对蛋白质晶体的损伤程度,但面对于微小晶体而言,辐射损伤问题更为突出。此外,低温冷冻技术也可能会导致一些精细结构信息丢失。这是目前传统的同步辐射晶体学所面临的问题。近年来,串行晶体学作为一种解析蛋白质晶体结构的新方法,其拥有室温采集,辐射损伤低,时间分辨等优势而得到广泛关注,而它的快速发展得益于自由电子激光的出现,探测器技术的发展,上样方式的创新以及数据处理方法的突破。基于自由电子激光的串行飞秒晶体学首次成功地在室温条件下获取蛋白质微小晶体的无损伤衍射数据,并解析了其高分辨结构,这促使着同步辐射串行晶体学研究开始蓬勃发展。为了充分发挥同步辐射已有的丰富实验资源和特点,解决同步辐射晶体学在微小晶体解析上的发展瓶颈,研究人员开始引入串行晶体学的方法开展相关研究,采用不同的上样方式证明了同步辐射串行晶体学的可行性,不仅解决室温下微小晶体的辐射损伤限制,同时具有微秒时间尺度上开展结构生物动态学研究的潜力。因此,本文为发挥上海光源BL17U1生物大分子线站的平台优势,在国内首次进行同步辐射串行晶体学上样技术的理论及实验研究,主要采取静电纺丝上样技术输运微小晶体,通过电压和流量的调控获得稳定的微小射流,并结合同轴静电纺丝和真空技术,搭配合适的子液缓解真空下样品喷射过程中结冰凝结的现象,延长了实验时间且避免了样品带电的潜在问题,解决了其在同步辐射上的实际应用;同时采用该方法采集了溶菌酶微小晶体的衍射数据,并对大量原始衍射图做初步的前处理以筛选有效命中的图像,探讨了同步辐射串行晶体学在微秒时间尺度上的发展潜力;然而受制于探测器性能和光强密度等硬件原因,目前不能较好地匹配静电纺丝较快的流速,导致其采集效率和数据信噪比较低,不适合做进一步的结构解析,但实验证明了其在同步辐射上应用的可行性,为未来同步辐射线站技术的升级改造提供了有价值的参考。
[Abstract]:Synchrotron radiation X-ray diffraction (SRXD) is the most important method to determine the three-dimensional structure of proteins. It can obtain high quality diffraction data of protein crystals. However, high brightness synchrotron radiation will cause radiation damage to protein crystals, resulting in the weakening and change of structural information. Although cryopreservation technology can reduce the damage degree of protein crystals caused by high intensity X-ray beams, the radiation damage is more serious in the face of small crystals. In addition, cryopreservation may result in the loss of fine structure information. This is the problem of traditional synchrotron radiation crystallography. In recent years, serial crystallography, as a new method for protein crystal structure analysis, has attracted much attention due to its advantages of room temperature acquisition, low radiation damage and time resolution. The rapid development of serial crystallography has benefited from the emergence of free electron lasers. The development of detector technology, the innovation of sample method and the breakthrough of data processing method. Serial femtosecond crystallography based on free electron laser successfully obtained the nondestructive diffraction data of protein microcrystals at room temperature for the first time, and analyzed their high-resolution structures. This led to the rapid development of serial crystallography of synchrotron radiation. In order to give full play to the abundant experimental resources and characteristics of synchrotron radiation and to solve the bottleneck in the development of synchrotron radiation crystallography in microcrystal resolution, researchers began to introduce serial crystallography to carry out related research. The feasibility of serial synchrotron radiation crystallography is proved by using different sampling methods. It not only solves the radiation damage limitation of microcrystals at room temperature, but also has the potential to study the structural dynamics on a microsecond time scale. Therefore, in order to give full play to the platform advantage of Shanghai Light Source BL17U1 Biomacromolecule Line Station, the theoretical and experimental study of synchrotron radiation serial crystallographic sampling technology is carried out in China for the first time. A stable micro jet was obtained by regulating the voltage and flow rate, and combined with coaxial electrostatic spinning and vacuum technology, suitable sub-liquid was used to alleviate the phenomenon of freezing and condensation during the spray process of the sample under vacuum. The experimental time is prolonged and the potential problem of the sample charged is avoided, and its practical application in synchrotron radiation is solved. Meanwhile, the diffraction data of microcrystals of lysozyme are collected by this method. A large number of original diffraction patterns are preprocessed to screen the effective hit images, and the development potential of synchrotron radiation serial crystallography in microsecond time scale is discussed. However, due to the performance of the detector and the intensity density of the detector and other hardware reasons, At present, the high velocity of electrostatic spinning can not be matched well, which leads to low acquisition efficiency and low signal-to-noise ratio of data, so it is not suitable for further structural analysis. However, the feasibility of its application in synchrotron radiation is proved by experiments. It provides a valuable reference for the upgrading of synchrotron radiation line station technology in the future.
【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.73

【参考文献】