Macromolecule Crystallization Screening Results Analysis

高分子结晶筛选结果分析

• 批准号: 9199371
• 负责人: Marc Lee Pusey
• 金额: $ 48.98万
• 依托单位: IXPRESSGENES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9199371
• 关键词: Area; Automation; Behavior; Businesses; Catalytic Domain; Chemicals; Complex; Computer software; Contracts; Crystallization; Data; Data Analyses; Data Set; Development; Encapsulated; Excision; Experimental Designs; Failure; Formulation; Goals; Guidelines; Housing; Laboratories; Licensing; Link; Manuals; Manufacturer Name; Methods; Microscope; Outcome; Output; Pharmacologic Substance; Phase; Probability; Process; Proteins; Research; Roentgen Rays; Screening Result; Series; Services; Set protein; Source; Structure; Structure-Activity Relationship; Testing; Thermococcus; Time; United States National Institutes of Health; Vendor; Work; Writing; X-Ray Crystallography; analytical method; base; combinatorial; computer program; cost; data acquisition; design; exosome; file format; hyperthermophile; imaging system; improved; literature survey; macromolecule; meetings; microbial; pathogen; programs; protein structure; proton-translocating pyrophosphatase; research study; response; screening; software development; structural biology; success; tool

Project Summary Crystallization, followed by subsequent structure determination, is a major step in understanding the structure-function relationship of macromolecules. Understanding macromolecule structure has become a key part in the development of new pharmaceuticals, and is a major area of NIH research. Crystallization however is also the rate limiting step, despite technological efforts to automate the set-up and crystallization data acquisition processes. Macromolecule crystallization conditions are arrived at by screening experiments, where the target material is typically subjected to hundreds or even thousands of different chemical cocktails. In most cases screening experiments fail as they do not result in a crystal. We propose that screening experiments contain useful information about the target proteins behavior in response to the tested solution conditions. No screen or group of screens can systematically cover the combinatorial chemical space for protein crystallization, and we hypothesize that in the absence of clear positive hits scored results can be analyzed to determine these factors. The analysis method developed is called the Associated Experimental Design (AED) approach. The analysis identified the most significant factors and a 96 condition screen based on those factors is prepared for each protein and set up. In the (ongoing) Phase I effort the AED software is being progressively evolved, adding functions for aiding in prioritizing the screen factors employed for likely success in crystallization. The software is written to not duplicate input conditions for a given protein in the output; i.e., all output conditions are new combinations of high probability factors as determined from the analysis. The software has been tested with 23 proteins to date. Of the 5 proteins that did not give crystals upon initial screening, 2 gave crystals from screens developed on the basis of the AED analysis. Of the 18 remaining proteins, 72% gave as many or more crystals in the single AED based screen than were obtained in the 4 x 96 condition screens. One of these 18 proteins was the RrP41-RrP42 archaeal exosome catalytic core complex. Based on the Phase I results the AED method shows considerable promise. A major advantage of this approach is that it fits into existing practice, making use of existing materials, methods, and data routinely generated in crystallization screening. The AED software can be used with any imaging system that gives a scored assessment of the results for each trial, including manual scoring by a user with a simple low power microscope. The Phase I results also showed that it can be used with a reduced, more granular, scoring scale. Success with this approach will increase the number of hits generated and greatly reduce the time and effort required for macromolecule crystallization. The proposed Phase II effort is to build upon the successful approach developed in Phase I and further develop the analytical methods employed.

项目摘要 结晶，随后是随后的结构测定，是理解晶体结构的主要步骤。 大分子结构-功能关系了解大分子结构已经成为 参与新药开发，是NIH研究的一个主要领域。然而，结晶 也是速率限制步骤，尽管技术上努力使设置和结晶数据自动化 收购过程。 通过筛选实验得到了高分子结晶条件， 材料通常经受数百甚至数千种不同的化学混合物。在大多数情况下 筛选实验失败，因为它们没有产生晶体。我们建议筛选实验包含 关于靶蛋白响应于测试溶液条件的行为的有用信息。没有屏幕 或一组筛网可以系统地覆盖用于蛋白质结晶的组合化学空间， 我们假设，在没有明确的阳性命中的情况下，可以分析评分结果来确定这些 因素开发的分析方法被称为关联实验设计（AED）方法。的 分析确定了最重要的因素，并根据这些因素准备了96个条件筛选， 每种蛋白质和设置。在（正在进行的）第一阶段工作中，AED软件正在逐步发展， 增加了用于帮助优先化可能成功结晶所采用的筛选因素的功能。的 编写软件以不在输出中复制给定蛋白质的输入条件;即，所有输出条件 是根据分析确定的高概率因素的新组合。该软件已经 到目前为止，已经测试了23种蛋白质。在初始筛选时不产生晶体的5种蛋白质中，2种产生晶体。 晶体从屏幕上开发的AED分析的基础上。在剩下的18种蛋白质中，72%的蛋白质为 在基于AED的单一筛选器中获得的晶体比在4 × 96条件筛选器中获得的晶体多或多。 这18种蛋白质之一是RrP 41-RrP 42古细菌外泌体催化核心复合物。 基于第一阶段的结果，AED方法显示出相当大的前景。这样做的一个主要优点是 一种方法是，它适合现有的做法，利用现有的材料，方法和数据例行 在结晶筛选中生成。AED软件可以与任何成像系统一起使用， 每次试验结果的评分评估，包括用户使用简单的低功效手动评分 显微镜第一阶段的结果还表明，它可以与一个减少，更细粒度，评分量表。 这种方法的成功将增加生成的点击数，并大大减少时间和精力 高分子结晶所需的。拟议的第二阶段工作是建立在成功的 第一阶段开发的方法，并进一步发展所采用的分析方法。