STRUCTURAL ANALYSIS OF MACROMOLECULAR COMPLEX

大分子复合物的结构分析

• 批准号: 6424722
• 负责人: JACQUELINE MILNE
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6424722
• 关键词: X ray crystallography; biological signal transduction; cell biology; electron microscopy; enzyme complex; macromolecule; membrane proteins; physical model; protein biosynthesis; protein structure; pyruvate dehydrogenase; structural biology

An outstanding challenge in cell biology is to define at atomic resolution, the molecular events that occur at biological membranes such as the transduction of extracellular signals into intracellular responses, energy conversion, and the generation or transport of small molecules and ions. A full understanding of these processes will require atomic resolution structures of membrane proteins trapped in their basal and activated states, as well as atomic views of multi-protein complexes that nucleate at cellular membranes. A major focus of my laboratory is the structure determination of such large multiprotein assemblies by analysis of high resolution images of single particles. In this method, large numbers of images of individual protein molecules are recorded using very low electron doses, sorted into distinct classes, and then added together to obtain distinct views of the molecule that have a high signal to noise ratio. The averaged views are then oriented with respect to each other, and used to reconstruct a model of the three-dimensional structure. The pace of recent progress in this field suggests that this approach of crystallography without crystals may have great potential to probe biologically-relevant complexes that are too large to be analyzed by NMR methods or that do not crystallize easily in the two- or three-dimensional arrays required for electron microscopic or X-ray crystallographic studies. The catalytic core of the pyruvate dehydrogenase is an excellent model system for refinement of single particle methods. Sixty copies of this enzyme, E2CD, assemble into a 1,800 kDa icosahedral complex that is readily purified and that shows a number of distinct orientations on electron micrographs. We are optimizing methods to accurately orient the single molecules and to correct distortions introduced during image collection on the electron microscope. Currently, processing of 4500 individual molecular images has led to a three-dimensional model that has a resolution of 14 +. The predicted envelope of this structure is in excellent agreement with the recently determined X-ray structure. Analysis of the catalytic core when it is complexed to the accessory enzymes E1 or E3 has yielded a three-dimensional model that has a resolution of 35 +. The study of such complexes is useful even at modest resolution since three dimensional structures of the individual components solved by X-ray crystallography can be docked into density maps determined by electron microscopy. Refinement of these methods will also be useful for the analysis of human P-glycoprotein and for related structural projects of other membrane proteins currently underway in the laboratory.

细胞生物学中的一个突出挑战是以原子分辨率定义发生在生物膜上的分子事件，例如细胞外信号到细胞内反应的转导、能量转换以及小分子和离子的产生或运输。 这些过程的全面理解将需要被困在其基础和激活状态的膜蛋白的原子分辨率结构，以及在细胞膜上成核的多蛋白质复合物的原子视图。 我的实验室的一个主要重点是通过分析单粒子的高分辨率图像来确定这种大型多蛋白质组装体的结构。 在这种方法中，使用非常低的电子剂量记录单个蛋白质分子的大量图像，将其分类为不同的类别，然后将其加在一起以获得具有高信噪比的分子的不同视图。 平均视图然后相对于彼此定向，并且用于重建三维结构的模型。 在这一领域的最新进展的步伐表明，这种方法的晶体学没有晶体可能有很大的潜力来探测生物相关的复合物太大，不能通过NMR方法分析，或不容易结晶的二维或三维阵列所需的电子显微镜或X射线晶体学研究。 丙酮酸脱氢酶的催化核心是一个很好的模型系统，用于改进单粒子方法。这种酶的60个拷贝，E2 CD，组装成一个1,800 kDa的二十面体复合物，很容易纯化，并显示了一些不同的方向上的电子显微镜照片。 我们正在优化方法，以准确地定位单个分子，并纠正电子显微镜图像收集过程中引入的失真。 目前，4500个单独的分子图像的处理已经产生了具有14 +分辨率的三维模型。 该结构的预测包络与最近确定的X射线结构非常一致。 当催化核心与辅助酶E1或E3复合时，对催化核心的分析产生了分辨率为35 +的三维模型。 这种复合物的研究是有用的，即使在适度的分辨率，因为三维结构的各个组件解决了X射线晶体学可以对接到密度图确定的电子显微镜。 这些方法的改进也将有助于分析人类P-糖蛋白和其他膜蛋白的相关结构项目目前正在实验室进行。