Technology Development for High-Throughput Biomolecular CryoSAXS

项目摘要/摘要 在过去的15年里，小角X射线散射(SAXS)已经成为学术界的一种重要工具。 政府，以及商业研究和开发，以探索蛋白质的结构和功能， 核酸和大分子复合体。SAXS提供精确但低分辨率的结构信息 从溶液中的生物分子和络合物，而不需要结晶或标记生物分子，无论 指分子量或分子有序性的程度。先进且易于使用的数据分析套件支持 快速解释SAXS剖面，产生从分子旋转半径到结构的信息 信封和套装。大多数同步加速器源都有专用的BioSAXS束线，允许 每天测量数百个样本，但正在进行的提高其吞吐量的努力并没有 与用户需求的快速扩张保持同步。 目前，所有的BioSAXS都是在室温或接近室温的情况下对样品进行的。由于生物分子 初始表达/纯化、SAXS测量和辐射之间的聚集和降解 由于受到照明性X射线的破坏，每次测量需要大量样品。样本单元必须 在每次测量之间加载，然后彻底清洗，因此数据收集占空比非常低。 大样本量要求、低测量占空比和高用户需求是关键的瓶颈 在不断扩大的BioSAXS中，特别是在高通量参数和配基相互作用方面 筛选和研究难以产生的蛋白质或复合体，生物SAXS可能具有的应用 对人类健康影响最大的。 在我们最近的演示实验的基础上，这个项目旨在开发技术和方法 用于在低温下对生物分子样品进行高通量SAXS。就像冰晶学一样， CryoSAXS每次测量需要的样品体积应该小得多，允许在 净化后立即在家中实验室，轻松存储和运输样品，以及自动化的高通量数据 占空比接近50%的集合。这将大大提高两者的使用效率 生物分子和同步加速器束时间，大大扩展了BioSAXS研究的潜在范围。钥匙 要开发的这项技术的方面是(1)固定和可重现的路径长度样品池和样品 能够快速、均匀地冷却样品的电池阵列；(2)用于样品冷却、储存、运输和 基于高通量结晶学的高通量处理；(3)对比度屏幕- 最大化已知低温电子密度的低温保护缓冲器，使热机械应力最小化； (4)基于测量的低温水化层模型，用于解释 (5)将该技术扩展到过冷液体中生物分子的SAXS研究 温度在~200至270 K之间的缓冲液。

Project Summary/Abstract In the last fifteen years, small-angle X-ray scattering (SAXS) has become a key tool in academic, government, and commercial research and development for probing the structure and function of proteins, nucleic acids, and macromolecular complexes. SAXS yields precise but low resolution structural information from biomolecules and complexes in solution, without the need to crystallize or label the biomolecule, regardless of molecular weight or the extent of molecular order. Sophisticated and easy-to-use data analysis suites enable rapid interpretation of SAXS profiles, yielding information ranging from molecular radius of gyration to structural envelopes and ensembles. Most synchrotron sources have dedicated bioSAXS beam lines that allow measurement of hundreds of samples per day, but ongoing efforts to improve their throughput have not kept pace with the rapid expansion in user demand. Currently, all bioSAXS is performed on samples at or near room temperature. Due to biomolecular aggregation and degradation between initial expression/purification and SAXS measurements and to radiation damage by the illuminating X-rays, large volumes of sample are required per measurement. Sample cells must be loaded and then thoroughly cleaned between each measurement, so data collection duty cycles are very low. Large sample volume requirements, low measurement duty cycles, and high user demand are critical bottlenecks in the continued expansion of bioSAXS, especially for high-throughput parameter and ligand interaction screening and for study of difficult to produce proteins or complexes, applications in which bioSAXS may have the greatest impact on human health. Building upon our recent demonstration experiments, this project aims to develop technology and methods for high-throughput SAXS on biomolecular samples at cryogenic temperatures. As with cryocrystallography, cryoSAXS should require much smaller sample volumes per measurement, allow sample preparation in the home lab immediately after purification, easy sample storage and shipping, and automated high-throughput data collection with duty cycles approaching 50%. This will enable dramatically more efficient use of both biomolecules and synchrotron beam time, and significantly expand the potential scope of bioSAXS studies. Key aspects of this technology to be developed are (1) fixed and reproducible path length sample cells and sample cell arrays that enable rapid, homogeneous sample cooling; (2) tools for sample cooling, storage, shipment, and high-throughput handling based on those for high-throughput cryocrystallography; (3) screens of contrast- maximizing cryoprotective buffers of known cryogenic electron density that minimize thermomechanical stresses; (4) measurement-based modeling of hydration layers at cryogenic temperatures, needed for interpretation of cryoSAXS profiles; and (5) extensions of this technology to SAXS studies on biomolecules in supercooled liquid buffers at temperatures between ~200 and 270 K.