Small angle x-ray scattering studies of biological macromolecules at cryogenic temperatures

蛋白质的生物学功能与其形状有关，因此可以通过测量其结构以及当蛋白质与其他分子相互作用时该结构如何变化来了解蛋白质的功能。这项任务具有挑战性，因为许多蛋白质都很小，直径比人类头发的宽度小一万倍。结构生物学领域依赖于利用实验方法，在如此小的长度尺度上提供准确的结构信息。在过去的十年中，由于国家设施（同步辐射源）的高强度X射线束的可用性以及返回结构包络的复杂分析程序的开发，小角X射线散射（SAXS）已成为快速获得蛋白质和其他大生物分子结构的可靠工具。SAXS的最大优点是它能够报告溶液中生物大分子的结构，而不需要专门的样品制备。蛋白质的形状现在可以用一种简单而直接的方法来确定。然而，用于测量样品的辐射也会损坏样品，改变其结构。这个问题在其他基于辐射的方法中也遇到过，包括X射线晶体学和电子显微镜。对于后者，使用低温冷冻已将损害降低到可容忍的水平：生物样品在~100 K的温度下免受辐射。本项目的目标是设计和建造两种仪器，将准备快速冷冻样品优化SAXS实验：冷冻SAXS。由于冷冻样品可以耐受更长时间的辐射暴露，而无需担心辐射引起的结构变化，因此这种方法将允许从即使少量的珍贵样品中获得结构。低温SAXS的成功演示将在大型结构生物学社区中引起巨大的兴趣。本项目产生的所有计划/设计将通过期刊出版、会议演示、在现有的晶体学和SAXS公共论坛上发布以及网络研讨会等方式公开。通过使用同步加速器辐射源进行传播也极为有效，因为大量用户通过用户会议、设施网站和讲习班了解新技术。该项目的一个长期目标是开发一个工具包，使结构生物学社区很容易获得这项技术。

A protein's biological job is related to its shape, thus insight can be gained into a protein's function by measuring its structure and how this structure changes as the protein interacts with other molecules. This task is challenging because many proteins are small, with diameters about ten thousand times less than the width of a human hair. The field of structural biology relies on exploiting experimental methods that provide accurate structural information on such small length scales. In the last decade, thanks to the availability of high intensity x-ray beams at national facilities (synchrotron sources) as well as the development of sophisticated analysis programs that return structural envelopes, small angle x-ray scattering (SAXS) has become a reliable tool for rapidly obtaining structures of proteins and other large biological molecules. The greatest strength of SAXS is its ability to report structures of biological macromolecules insolution, without the need for specialized sample preparation. A protein's shape can now be determined in a simple and straightforward way. However, the radiation used to measure the sample can also damage it, changing its structure. This problem has been encountered in other radiation-based methods, including x-ray crystallography and electron microscopy. For these latter fields, the use of cryo-freezing has reduced damage to tolerable levels: biological samples are protected from radiation at temperatures of ~100 K. The goal of this project is to design and construct two instruments that will prepare flash-frozen samples optimized for SAXS experiments: cryo-SAXS. Because frozen samples can tolerate longer exposure to radiation, without concern for radiation-induced structural changes, this method will allow structures to be obtained from even small amounts of precious sample. The successful demonstration of cryo-SAXS will garner tremendous interest within the large structural biology community. All plans/designs generated as a result of this project will be made public, through journal publication, presentations at conferences, posting on existing public forums for crystallography and SAXS, and webinars. Dissemination through use at synchrotron sources is also extremely effective, as a large number of users pass through and are exposed to new technologies through users' meetings, facility websites and workshops. A long-term goal of this project is to develop a kit that makes this technology easily available to the structural biology community.