Fluorescence Intensity-based Scoring of Macromolecule Crystallization Plates
基于荧光强度的高分子结晶板评分
基本信息
- 批准号:8452862
- 负责人:
- 金额:$ 37.5万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2010
- 资助国家:美国
- 起止时间:2010-07-12 至 2015-03-31
- 项目状态:已结题
- 来源:
- 关键词:AgreementAlgorithmsAreaAutomationBedsBindingCollectionComplexComputer softwareCrystallizationDataData AnalysesDatabasesDevelopmentDiseaseDrug TargetingEyeFailureFluorescenceFluorescence MicroscopyFluorescent ProbesGoalsHandHousingImageImage AnalysisIntegral Membrane ProteinLabelLaboratoriesLeadLightManualsMediationMethodologyMethodsNeedlesOutcomeOutputPerformancePharmacologic SubstancePhasePrecipitationPriceProceduresProcessProteinsRelative (related person)ReporterResearchRoentgen RaysShapesSoftware DesignSolutionsSpottingsStagingStructureStructure-Activity RelationshipSystemTechnologyTestingTimeUnited States National Institutes of HealthWorkX-Ray Crystallographybasecommercializationcostdata acquisitiondesignfluorescence imagingimage processingimprovedinstrumentinterestmacromoleculemethod developmentmolecular dynamicsprotein structurepublic health relevanceresponsescreeningsoftware developmentstructural biologysuccesstool
项目摘要
DESCRIPTION (provided by applicant): 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. The Phase I effort successfully demonstrated that a low cost epifluorescence type crystallization plate imaging system could be assembled, and that lead crystallization conditions could be obtained from apparently failed outcomes by intensity analysis of the fluorescence images. The method is based upon trace covalent labeling, defined as < 0.5% of the molecules being labeled, of the protein using a fluorescent probe that excites and emits in the visible spectrum. The major objectives of this proposal are to expand upon the Phase I results. First is the improvement of the software for the rapid scoring of the crystallization screen images, to expand that capability to include scoring for different crystallization outcomes (needle, plate, 3D crystal), and to further improve the scoring success rate. Second is the implementation of a multicolor fluorescence capability to make the instrument more suitable for the crystallization of macromolecule complexes, followed by the testing and demonstration of that capability. Third is to develop a labeling methodology suitable for use with integral membrane proteins. Fourth is to further refine the instrument and methods by continued use and testing in our laboratory. Experimentally, trace fluorescently labeled protein will be subjected to crystallization screens and the outcomes periodically imaged. Intensity-based image analysis, using the evolving software as it is developed during the proposal period, will be carried out. Precipitated conditions which show suitable scores based on the image analysis will be subjected to optimization screening, and based upon the Phase I effort results we expect a correlation between the scores obtained and subsequent crystallization. Previous research has shown that fluorescence can be a powerful aid in finding and identifying crystals in screening plates (Judge et al., 2005; Forsythe et al., 2006; Groves et al., 2007; Pusey et al., 2008). Crystallization gives the most densely packed state for a protein, and therefore trace fluorescently labeled protein will have the greatest fluorescence intensity relative to clear or precipitated outcomes. The covalently bound probe serves as a reporter to the protein's response to the solution conditions. Some precipitates showed 'bright spots' of fluorescence, and many of these outcomes were subsequently be optimized to crystallization conditions (Pusey et al., 2008; Phase I results). Thus intensity-based scoring of precipitation outcomes may be used to discriminate between non-productive and potentially productive precipitation results. Fluorescence intensity-based crystallization screen scoring is found to be fast, with image processing times currently 3 seconds.
描述(申请人提供):结晶,随后的结构测定,是理解大分子的结构-功能关系的主要步骤。了解大分子结构已成为新药开发的关键部分,也是美国国立卫生研究院研究的主要领域。然而,结晶也是速度限制步骤,尽管技术努力使设置和结晶数据采集过程自动化。第一阶段的工作成功地证明了一种低成本的荧光型结晶板成像系统可以组装起来,并且通过荧光图像的强度分析可以从明显失败的结果中获得铅的结晶条件。该方法基于微量共价标记,定义为使用在可见光谱中激发和发射的荧光探针标记蛋白质的<;0.5%的分子。这项提案的主要目标是扩大第一阶段的成果。首先是改进结晶屏幕图像快速评分的软件,扩大这一能力,包括对不同结晶结果(针、板、3D晶体)的评分,并进一步提高评分成功率。第二是实现多色荧光能力,使该仪器更适合于大分子络合物的结晶,随后对该能力进行测试和演示。第三是开发一种适用于完整膜蛋白的标记方法。四是通过在实验室的继续使用和测试,进一步细化仪器和方法。实验上,微量荧光标记的蛋白质将经过结晶筛查,结果将定期成像。将使用提议期间开发的不断演变的软件,进行基于强度的图像分析。根据图像分析显示适当分数的沉淀条件将接受优化筛选,并且基于第一阶段的努力结果,我们期望获得的分数与后续结晶之间的关联。先前的研究表明,荧光可以有力地帮助发现和识别筛板中的晶体(贾奇等人,2005年;Forsythe等人,2006年;Groves等人,2007年;Pusey等人,2008年)。结晶为蛋白质提供了最密集的堆积状态,因此,相对于透明或沉淀的结果,微量荧光标记的蛋白质将具有最大的荧光强度。共价结合的探针作为蛋白质对溶液条件的反应的报告。一些沉淀物显示出荧光的“亮点”,其中许多结果随后被优化为结晶条件(Pusey等人,2008年;第一阶段结果)。因此,可使用基于强度的降水结果评分来区分非生产性和潜在生产性降水结果。基于荧光强度的结晶屏幕评分被发现是快速的,图像处理时间目前为3秒。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Marc Lee Pusey其他文献
Marc Lee Pusey的其他文献
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{{ truncateString('Marc Lee Pusey', 18)}}的其他基金
Macromolecule Crystallization Screening Results Analysis
高分子结晶筛选结果分析
- 批准号:
9353835 - 财政年份:2015
- 资助金额:
$ 37.5万 - 项目类别:
Macromolecule Crystallization Screening Results Analysis
高分子结晶筛选结果分析
- 批准号:
9199371 - 财政年份:2015
- 资助金额:
$ 37.5万 - 项目类别:
Fluorescence Intensity-based Scoring of Macromolecule Crystallization Plates
基于荧光强度的高分子结晶板评分
- 批准号:
8642652 - 财政年份:2010
- 资助金额:
$ 37.5万 - 项目类别:
Fluorescence Intensity-Based Scoring of Macromolecule Crystallization Plates
基于荧光强度的高分子结晶板评分
- 批准号:
7801742 - 财政年份:2010
- 资助金额:
$ 37.5万 - 项目类别:
Fluorescence Anisotropy-based Macromolecule Crystallization Screening
基于荧光各向异性的高分子结晶筛选
- 批准号:
7998996 - 财政年份:2008
- 资助金额:
$ 37.5万 - 项目类别:
Fluorescence Anisotropy-based Macromolecule Crystallization Screening
基于荧光各向异性的高分子结晶筛选
- 批准号:
8139679 - 财政年份:2008
- 资助金额:
$ 37.5万 - 项目类别:
Fluorescence Anisotropy-based Macromolecule Crystallization Screening
基于荧光各向异性的高分子结晶筛选
- 批准号:
7479549 - 财政年份:2008
- 资助金额:
$ 37.5万 - 项目类别:
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