Solving the Modulated Structures in Profilin:Actin Crystals

解析 Profilin:肌动蛋白晶体中的调制结构

• 批准号: 0718661
• 负责人: Gloria Borgstahl
• 金额: $ 50万
• 依托单位: University of Nebraska Medical Center
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0718661&HistoricalAwards=false
• 关键词: Solving; Modulated; Structures; Profilin; Actin

Modulated crystals are often encountered in crystallography. Methods for their structural characterization need further development. This research is focused on the structure determination of modulated crystals of profilin:actin. The objectives of this research are two-fold; first, to continue to develop new macromolecular X-ray crystallographic methods for the structure solution of aperiodic, modulated crystals, and second, to test the hypothetical model that the actin ''ribbon'' found in PA crystals represents a structural intermediate of filamentous actin. This research will employ protein crystal growth, X-ray diffraction data collection and structure determination. At several points during the project, innovative approaches to protein crystallography and software will be developed. The unique diffraction pattern from the modulated crystals contains unusual off-lattice satellite reflections next to the main reflections. Strategies for collecting X-ray diffraction data that detect and separate satellite reflections from the main reflections will be developed. Downstream of data collection, existing software from chemical (AKA small molecule) crystallography will be adapted and used to process the modulated diffraction pattern. From the diffraction data, the type and direction of the modulation(s) will be determined. Then additional chemical crystallography software will be extensively modified and combined with macromolecular algorithms to solve the modulated crystallographic phase problem, produce multidimensional electron density images and refine the modulated atomic structure against the experimental diffraction data. Finally, the resulting structure(s) will be analyzed within the framework of the current cell biology literature. It is anticipated that these structural results will test the validity of the ribbon-to-helix model of actin filament formation. Broader ImpactsResearch will be integrated with education through partnership with the University of Nebraska-Omaha to develop undergraduate research internships and coursework in protein purification and crystallization with profilin:actin as a central theme; the formation of ''Graduate Student Training Program in Structural Biology and Biophysics'' at UNMC; supporting national conference workshops on how to solve modulated structures for the continued education of chemical and macromolecular crystallographers at all levels; and the incorporation of research results and methods into graduate-level coursework. The PI's laboratory provides a diverse and multicultural environment for the participation of underrepresented students at all levels of training in this research program.

调制晶体在晶体学中经常遇到。其结构表征的方法需要进一步发展。本研究的重点是对profilin：actin的调制晶体的结构测定。本研究的目标是双重的，第一，继续发展新的大分子X射线晶体学方法的非周期性的，调制晶体的结构解决方案，第二，测试的假设模型，在PA晶体中发现的肌动蛋白“丝带”代表丝状肌动蛋白的结构中间体。这项研究将采用蛋白质晶体生长，X射线衍射数据收集和结构测定。在项目期间的几个点上，将开发蛋白质晶体学和软件的创新方法。来自调制晶体的独特衍射图案在主反射旁边包含不寻常的非晶格卫星反射。将制定收集X射线衍射数据的战略，以探测卫星反射并将其与主要反射分开。在数据收集的下游，将调整现有的化学（AKA小分子）晶体学软件，并用于处理调制衍射图。根据衍射数据，将确定调制的类型和方向。然后，额外的化学晶体学软件将被广泛修改，并与大分子算法相结合，以解决调制晶体学相位问题，产生多维电子密度图像，并根据实验衍射数据细化调制原子结构。最后，将在当前细胞生物学文献的框架内分析所得结构。预计这些结构的结果将测试的有效性的肌动蛋白丝形成的带状到螺旋模型。更广泛的影响研究将通过与内布拉斯加大学奥马哈分校的伙伴关系与教育相结合，以profilin：actin为中心主题，开发蛋白质纯化和结晶方面的本科生研究实习和课程;在UNMC形成“结构生物学和生物制药学研究生培训计划”;支持关于如何解决调制结构问题的国家会议讲习班，以便对各级化学和大分子晶体学家进行继续教育;以及将研究成果和方法纳入研究生课程。PI的实验室为代表性不足的学生参与该研究项目的各级培训提供了多元化和多元文化的环境。