High-throughput SAXS for the characterization of biomolecules in solution: a practical approach.
High-throughput SAXS for the characterization of biomolecules in solution: a practical approach.
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DOI：
10.1007/978-1-62703-691-7_18
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Hura, Greg L
中科院分区：
文献类型：
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作者：
Dyer, Kevin N;Hammel, Michal;Rambo, Robert P;Tsutakawa, Susan E;Rodic, Ivan;Classen, Scott;Tainer, John A;Hura, Greg L
最近的创新，收集X射线散射的解决方案，含有纯化的大分子在高通量尚未真正利用的生物界。然而，鉴于序列和基因组学数据的增长速度明显超过结构生物学结果，这种能力变得至关重要。鉴于序列和结构方法之间信息增长率的巨大不匹配，它们的高通量和高成功率使得高通量小角X射线散射（HT-SAXS）分析越来越有价值。HT-SAXS通过定义控制细胞生物学的灵活和动态复合物，将序列以及NMR和晶体学结果与生物学结果联系起来。通常属于bio-SAXS的保护伞下，HT-SAXS数据收集管道已经或正在开发中的大多数同步加速器。研究人员如何将他们感兴趣的生物分子实际上进入这些管道，平衡样品要求和管理HT-SAXS数据输出格式因设施而异。虽然这些特征不太可能在同步加速器光束线上标准化，但对一条管道的HT-SAXS问题的详细描述为研究人员提供了他们将遇到的一般程序的实用指南。运行时间最长且通常可访问的HT-SAXS终端站之一是位于加利福尼亚州伯克利的高级光源的SIBYLS光束线。在这里，我们描述了SIBYLS HT-SAXS管道的当前状态，研究人员需要整合到其中，输出格式和2年运行结果的摘要。对累积数据的评估反映了浓度、背景、缓冲液、样品处理、样品运输、均匀性要求、误差来源、聚集、辐射敏感性、解释和关注标志等问题。通过定量研究成功和失败的样本和数据特征的函数，我们定义的实际问题，注意事项和概念的最佳应用HT-SAXS技术的生物样品。
The recent innovation of collecting X-ray scattering from solutions containing purified macromolecules in high-throughput has yet to be truly exploited by the biological community. Yet, this capability is becoming critical given that the growth of sequence and genomics data is significantly outpacing structural biology results. Given the huge mismatch in information growth rates between sequence and structural methods, their combined high-throughput and high success rate make high-throughput small angle X-ray scattering (HT-SAXS) analyses increasingly valuable. HT-SAXS connects sequence as well as NMR and crystallographic results to biological outcomes by defining the flexible and dynamic complexes controlling cell biology. Commonly falling under the umbrella of bio-SAXS, HT-SAXS data collection pipelines have or are being developed at most synchrotrons. How investigators practically get their biomolecules of interest into these pipelines, balance sample requirements and manage HT-SAXS data output format varies from facility to facility. While these features are unlikely to be standardized across synchrotron beamlines, a detailed description of HT-SAXS issues for one pipeline provides investigators with a practical guide to the general procedures they will encounter. One of the longest running and generally accessible HT-SAXS endstations is the SIBYLS beamline at the Advanced Light Source in Berkeley CA. Here we describe the current state of the SIBYLS HT-SAXS pipeline, what is necessary for investigators to integrate into it, the output format and a summary of results from 2 years of operation. Assessment of accumulated data informs issues of concentration, background, buffers, sample handling, sample shipping, homogeneity requirements, error sources, aggregation, radiation sensitivity, interpretation, and flags for concern. By quantitatively examining success and failures as a function of sample and data characteristics, we define practical concerns, considerations, and concepts for optimally applying HT-SAXS techniques to biological samples.