本文選題：串行晶體學(xué) + 同步輻射　； 參考：《中國(guó)科學(xué)院研究生院(上海應(yīng)用物理研究所)》2017年碩士論文

【摘要】：同步輻射X射線衍射是當(dāng)前蛋白質(zhì)三維結(jié)構(gòu)測(cè)定的最主要方法,可高效獲取蛋白質(zhì)晶體的高質(zhì)量衍射數(shù)據(jù)。然而高亮度同步輻射對(duì)蛋白質(zhì)晶體會(huì)產(chǎn)生輻射損傷,導(dǎo)致其結(jié)構(gòu)信息減弱和改變。雖然晶體低溫冷凍技術(shù)可以降低高強(qiáng)度的X射線光束對(duì)蛋白質(zhì)晶體的損傷程度,但面對(duì)于微小晶體而言,輻射損傷問(wèn)題更為突出。此外,低溫冷凍技術(shù)也可能會(huì)導(dǎo)致一些精細(xì)結(jié)構(gòu)信息丟失。這是目前傳統(tǒng)的同步輻射晶體學(xué)所面臨的問(wèn)題。近年來(lái),串行晶體學(xué)作為一種解析蛋白質(zhì)晶體結(jié)構(gòu)的新方法,其擁有室溫采集,輻射損傷低,時(shí)間分辨等優(yōu)勢(shì)而得到廣泛關(guān)注,而它的快速發(fā)展得益于自由電子激光的出現(xiàn),探測(cè)器技術(shù)的發(fā)展,上樣方式的創(chuàng)新以及數(shù)據(jù)處理方法的突破。基于自由電子激光的串行飛秒晶體學(xué)首次成功地在室溫條件下獲取蛋白質(zhì)微小晶體的無(wú)損傷衍射數(shù)據(jù),并解析了其高分辨結(jié)構(gòu),這促使著同步輻射串行晶體學(xué)研究開(kāi)始蓬勃發(fā)展。為了充分發(fā)揮同步輻射已有的豐富實(shí)驗(yàn)資源和特點(diǎn),解決同步輻射晶體學(xué)在微小晶體解析上的發(fā)展瓶頸,研究人員開(kāi)始引入串行晶體學(xué)的方法開(kāi)展相關(guān)研究,采用不同的上樣方式證明了同步輻射串行晶體學(xué)的可行性,不僅解決室溫下微小晶體的輻射損傷限制,同時(shí)具有微秒時(shí)間尺度上開(kāi)展結(jié)構(gòu)生物動(dòng)態(tài)學(xué)研究的潛力。因此,本文為發(fā)揮上海光源BL17U1生物大分子線站的平臺(tái)優(yōu)勢(shì),在國(guó)內(nèi)首次進(jìn)行同步輻射串行晶體學(xué)上樣技術(shù)的理論及實(shí)驗(yàn)研究,主要采取靜電紡絲上樣技術(shù)輸運(yùn)微小晶體,通過(guò)電壓和流量的調(diào)控獲得穩(wěn)定的微小射流,并結(jié)合同軸靜電紡絲和真空技術(shù),搭配合適的子液緩解真空下樣品噴射過(guò)程中結(jié)冰凝結(jié)的現(xiàn)象,延長(zhǎng)了實(shí)驗(yàn)時(shí)間且避免了樣品帶電的潛在問(wèn)題,解決了其在同步輻射上的實(shí)際應(yīng)用;同時(shí)采用該方法采集了溶菌酶微小晶體的衍射數(shù)據(jù),并對(duì)大量原始衍射圖做初步的前處理以篩選有效命中的圖像,探討了同步輻射串行晶體學(xué)在微秒時(shí)間尺度上的發(fā)展?jié)摿?然而受制于探測(cè)器性能和光強(qiáng)密度等硬件原因,目前不能較好地匹配靜電紡絲較快的流速,導(dǎo)致其采集效率和數(shù)據(jù)信噪比較低,不適合做進(jìn)一步的結(jié)構(gòu)解析,但實(shí)驗(yàn)證明了其在同步輻射上應(yīng)用的可行性,為未來(lái)同步輻射線站技術(shù)的升級(jí)改造提供了有價(jià)值的參考。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(上海應(yīng)用物理研究所)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O629.73

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相關(guān)期刊論文 前1條

1 汪啟勝;郁峰;黃勝;孫波;張坤浩;劉科;王志軍;徐春艷;王思勝;楊利峰;潘強(qiáng)巖;李良;周歡;崔瑩;徐琴;Thomas Earnest;何建華;;The macromolecular crystallography beamline of SSRF[J];Nuclear Science and Techniques;2015年01期

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本文編號(hào)：1892955