Choi Kyung H, Morais Marc
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-0304, USA,
Methods Mol Biol. 2014;1138:241-52. doi: 10.1007/978-1-4939-0348-1_15.
Small-angle X-ray scattering (SAXS) is a powerful reemerging biophysical technique that can be used to directly analyze many properties related to the size and shape of a macromolecule in solution. For example, the radius of gyration and maximum diameter of a macromolecule can be readily extracted from SAXS data, as can information regarding how well folded a protein is. Similarly, the molecular weight of macromolecular complexes can be directly determined from the complex's scattering profile, providing insight into the oligomeric state and stoichiometry of the assembly. Furthermore, recently developed procedures for ab initio shape determination can provide low-resolution (~20 Å) molecular envelopes of proteins/complexes in their native state. In conjunction with high-resolution structural data, more sophisticated analysis of SAXS data can help address questions regarding conformational change, molecular flexibility, and populations of states within molecular ensembles. Because SAXS samples are easy to prepare and SAXS data is relatively easy to collect, the technique holds great promise for investigating the structure of macromolecules and their assemblies as well as monitoring and modeling their conformational changes. Here we describe typical steps in SAXS sample preparation and data collection and analysis and provide examples of SAXS analysis to investigate the structure and function of dengue virus NS3 and NS5.
小角X射线散射（SAXS）是一种强大且重新兴起的生物物理技术，可用于直接分析许多与溶液中大分子的大小和形状相关的性质。例如，大分子的回转半径和最大直径可以很容易地从小角X射线散射数据中提取出来，蛋白质折叠程度的相关信息也可以提取出来。同样，大分子复合物的分子量可以直接从复合物的散射图谱中确定，从而深入了解组装体的寡聚状态和化学计量。此外，最近开发的从头算形状确定程序可以提供处于天然状态的蛋白质/复合物的低分辨率（约20 Å）分子轮廓。结合高分辨率结构数据，对小角X射线散射数据进行更复杂的分析有助于解决有关构象变化、分子柔韧性以及分子集合中状态群体的问题。由于小角X射线散射样品易于制备，且小角X射线散射数据相对易于收集，该技术在研究大分子及其组装体的结构以及监测和模拟其构象变化方面具有很大的前景。在这里，我们描述小角X射线散射样品制备、数据收集和分析的典型步骤，并提供小角X射线散射分析的实例，以研究登革病毒NS3和NS5的结构和功能。
