Solving the Modulated Structures in Profilin:Actin Crystals

调制晶体是结晶学中经常遇到的现象。它们的结构表征方法还需要进一步发展。本研究主要研究Profilin：肌动蛋白调制晶体的结构测定。这项研究的目标有两个：第一，继续开发新的大分子X射线结晶学方法，用于非周期调制晶体的结构溶液；第二，验证在PA晶体中发现的肌动蛋白“带”代表丝状肌动蛋白结构中间体的假设模型。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。在项目期间的几个时间点上，将开发蛋白质结晶学和软件的创新方法。调制晶体的独特衍射图在主反射旁边包含不同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，来自化学(又名小分子)结晶学的现有软件将被改编并用于处理调制的衍射图。根据衍射数据，将确定调制的类型和方向(S)。然后，将对额外的化学结晶学软件进行广泛的修改，并将其与大分子算法相结合，以解决调制晶相问题，产生多维电子密度图像，并根据实验衍射数据精炼调制原子结构。最后，将在当前细胞生物学文献的框架内对得到的结构(S)进行分析。预计这些结构结果将检验肌动蛋白细丝形成的带状到螺旋模型的有效性。更广泛的影响通过与内布拉斯加-奥马哈大学合作，将研究与教育结合起来，开发蛋白质纯化和结晶方面的本科生研究实习和课程作业，以Profilin：肌动蛋白为中心主题；在UNMC形成“结构生物学和生物物理学研究生培训计划”；支持关于如何解决调制结构的全国会议讲习班，以供各级化学和高分子结晶学家继续教育；并将研究成果和方法纳入研究生课程。国际学生联合会的实验室提供了一个多样化和多元文化的环境，供未被充分代表的学生参加这项研究计划的所有级别的培训。

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.