Structural Analysis of Macromolecular Complexes by Elect

大分子配合物的结构分析

• 批准号: 7338519
• 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/7338519
• 关键词: Structural; Analysis; Macromolecular; Complexes; Elect

I. Molecular architecture of multi-protein enzyme complexes: We have been using cryo-electron microscopy to study the architecture of the pyruvate dehydrogenase complex as an example of a multi-enzyme complex involved in energy metabolism. So far, we have determined the three dimensional structures at 27 resolution for two giant icosahedral pyruvate dehydrogenase complexes from Bacillus stearothermophilus using electron cryo-microscopy: one is a 11 MDa complex composed of 60 copies of E1 and E2 enzymes, and the other is a 9 MDa complex composed of 60 copies of E2 and E3 enzymes. By positioning the previously determined structures of E1, E3 and the three domains of E2 into the model, we have arrived at atomic interpretations for the entire E1E2 and E2E3 complexes. To our knowledge, these are the largest non-viral protein complexes for which such atomic models are available, and they provide unique insights into the functional mechanism of a fascinating cellular machine that remains inaccessible to structural analysis by X-ray crystallography. In ongoing work, we are studying the role of flexible linker regions in maintaining the size and shape of the complex using a combination of electron microscopic and other biophysical approaches. Studies of mammalian pyruvate dehydrogenase complexes to understand the mechanisms underlying regulation by kinases and phosphatases are also planned. II. Spatial map of molecular complexes in healthy and diseased mitochondria We are using Dictyostelium discoideum, a genetically amenable haploid model system that undergoes a defined program of cellular differentiation and morphogenesis, to develop methods to determine the molecular architecture of mitochondria. Dictyostelium mitochodria are small ( 0.5-0.7 micrometer in diameter and 0.5-2.0 micrometer in length) and easily isolated, and can be imaged using electron tomography. We have established the feasibility of this imaging approach in our recent work on electron tomography of an intact microorganism Bdellovibrio bacteriovorus, where we have demonstrated that it is possible to visualize individual molecular machines in the context of an intact cell. In related experiments, we are imaging whole Dictyostelium cells using a newly developed 3D imaging tool known as dual beam microscopy to map the spatial distribution of mitochondria in cells at different stages of development, and in mitochondrial mutants. We expect these studies will be relevant for the exploration of structural aspects of mitochondrial defects in cancer.

I.多蛋白酶复合物的分子结构：我们一直在使用冷冻电子显微镜研究丙酮酸脱氢酶复合物的结构，作为参与能量代谢的多酶复合物的一个例子。迄今为止，我们已经用冷冻电镜在27分辨率下测定了两个来自嗜热脂肪芽孢杆菌的二十面体丙酮酸脱氢酶复合物的三维结构：一个是由60个拷贝的E_1和E_2酶组成的11 MDa复合物，另一个是由60个拷贝的E_2和E_3酶组成的9 MDa复合物。通过将先前确定的E1，E3和E2的三个结构域的结构定位到模型中，我们已经达到了整个E1 E2和E2 E3复合物的原子解释。据我们所知，这些是最大的非病毒蛋白质复合物，这种原子模型是可用的，他们提供了独特的见解，一个迷人的细胞机器的功能机制，仍然无法通过X射线晶体学结构分析。在正在进行的工作中，我们正在研究的作用，灵活的连接器区域保持复杂的大小和形状，使用电子显微镜和其他生物物理方法的组合。哺乳动物丙酮酸脱氢酶复合物的研究，以了解激酶和磷酸酶的调节机制也计划。二.健康和患病线粒体中分子复合物的空间图我们正在使用Dictyosteelium discoideum，一种遗传上适合的单倍体模型系统，经历了一个确定的细胞分化和形态发生程序，以开发确定线粒体分子结构的方法。Dictyosteelium mitochodria很小（直径为0.5-0.7微米，长度为0.5-2.0微米），易于分离，并且可以使用电子断层扫描成像。我们已经建立了这种成像方法的可行性，在我们最近的工作，电子断层扫描的一个完整的微生物噬菌蛭弧菌，在那里我们已经证明，这是可能的可视化的背景下，一个完整的细胞的单个分子机器。在相关的实验中，我们使用新开发的3D成像工具（称为双光束显微镜）对整个Dictyosteoblasts细胞进行成像，以绘制不同发育阶段细胞中线粒体的空间分布，以及线粒体突变体。我们希望这些研究将与探索癌症中线粒体缺陷的结构方面有关。