NOVEL SOLUTION SAXS GEOMETRY FOR HIGH THROUGHPUT PROTEIN STRUCTURE CHARACTERIZAT

用于高通量蛋白质结构表征的新解决方案 SAXS 几何结构

• 批准号: 7721936
• 负责人: MARC NIEBUHR
• 金额: $ 1.92万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721936
• 关键词: Air; Aliquot; Blood capillaries; Cells; Computer Retrieval of Information on Scientific Projects Database; Condition; Data; Data Collection; Development; Funding; Future; Grant; Ice; Institution; Length; Measurement; Methods; Muramidase; Needles; Numbers; Proteins; Range; Research; Research Personnel; Resources; Sampling; Screening procedure; Shapes; Solutions; Solvents; Source; Standards of Weights and Measures; Syringes; Testing; Time; United States National Institutes of Health; Water; Work; capillary; evaporation; novel; protein structure; size

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The development of a novel data collection method for high throughput solution SAXS studies has been initiated, and uses sample aliquots that are suspended in the air as a droplet at the tip of a syringe needle. This new geometry eliminates the windows that are required for conventional sample aliquot containers, minimizing the background scattering level and eliminating the need for cleaning sample cells between each measurement. The possible use of smaller sample volumes than those required for the standard sample containers would allow larger number of sample screening conditions, and characterization of proteins that are very hard to express or purify. Another benefit is the potential elimination of air bubbles, which can appear in the sample aliquot within a capillary cell during sample delivery or oscillation. We have tested 5 and 3 microliter sample aliquot sizes to evaluate the feasibility of this approach for lysozyme solution x-ray scattering. The initial effort focused on evaluating the effects of spherical droplet shape on scattering data and validity of intensity scaling using the transmitted beam intensity. The Blu-Ice controlled Hamilton dispenser allowed the reproducible measurement of small sample aliquots of identical volume. Both water and lysozyme scattering profiles recorded in droplets are essentially identical to those obtained in a capillary cell in the Q range 0.015 - 0.6 A-1, demonstrating that the droplet shape effect is not a major concern and that the standard intensity scaling method works satisfactorily well. However, gradual time-dependent decay in scattering intensities was observed in both cases at the % level over several minutes, likely caused by solvent (water) evaporation which shortens the x-ray path length. Future studies will involve refrigerating the syringe needle to minimize this effect.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 已经开始开发用于高通量溶液SAXS研究的新型数据收集方法，并使用悬浮在空气中的样品等分试样作为注射器针头尖端的液滴。这种新的几何结构消除了传统样品等分容器所需的窗口，最大限度地减少了背景散射水平，并消除了每次测量之间清洁样品池的需要。 可能使用比标准样品容器所需体积更小的样品体积，这将允许更大数量的样品筛选条件，并表征非常难以表达或纯化的蛋白质。另一个益处是潜在地消除气泡，气泡可能在样品递送或振荡期间出现在毛细管池内的样品等分试样中。 我们已经测试了5和3微升的样品等分试样的大小，以评估这种方法的可行性溶菌酶溶液的X射线散射。最初的努力集中在评估球形液滴形状对散射数据的影响和使用透射光束强度的强度缩放的有效性。Blu-Ice控制的汉密尔顿分配器允许重复测量相同体积的小样品等分试样。在液滴中记录的水和溶菌酶散射曲线与在Q范围为0.015 - 0.6 A-1的毛细管池中获得的那些基本相同，表明液滴形状效应不是主要问题，并且标准强度缩放方法工作得令人满意。然而，在这两种情况下，在几分钟内观察到散射强度的逐渐时间依赖性衰减，可能是由于溶剂（水）蒸发缩短了X射线路径长度。未来的研究将涉及冷冻注射器针头，以尽量减少这种影响。