European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany.
Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.
Nat Protoc. 2016 Nov;11(11):2122-2153. doi: 10.1038/nprot.2016.113. Epub 2016 Oct 6.
Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis, so it is essential that the samples be pure and monodisperse for the duration of the experiment. This protocol outlines the basic physics of SAXS and SANS, and it reveals how the underlying conceptual principles of the techniques ultimately 'translate' into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size-exclusion chromatography (SEC) and light scattering. Also included are procedures that are specific to X-rays (in-line SEC-SAXS) and neutrons, specifically preparing samples for contrast matching or variation experiments and deuterium labeling of proteins.
小角度 X 射线散射 (SAXS) 和小角度中子散射 (SANS) 是用于提取结构参数并确定生物大分子、复合物和组装体在溶液中的整体结构和形状的技术。从样品中测量的散射强度包含来自照射样品体积内所有原子的贡献，包括溶剂和缓冲成分以及感兴趣的大分子。为了获得结构信息，必须制备完全匹配的溶剂空白，以便可以从样品散射中准确减去背景散射贡献，从而获得样品中大分子的净散射。此外，由污染物、聚集体、不匹配的溶剂、辐射损伤或其他因素引起的样品不均匀性会严重影响和复杂化数据分析，因此在实验过程中，样品必须是纯的和单分散的。该方案概述了 SAXS 和 SANS 的基本物理原理，并揭示了这些技术的基本概念原理如何最终“转化”为散射实验用样品制备的实际实验室指南，这些样品的质量足够高。该程序描述了如何使用凝胶电泳、尺寸排阻色谱 (SEC) 和光散射来制备和表征 SAXS 和 SANS 的蛋白质和核酸样品。还包括特定于 X 射线（在线 SEC-SAXS）和中子的程序，具体涉及为对比匹配或变化实验以及蛋白质氘标记制备样品。
