Macromolecular Complex Structure /High Res. Electron Mic

高分子复杂结构/高分辨率。

• 批准号: 6559266
• 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/6559266
• 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

Complex cellular processes such as signal transduction, gene expression, motility and energy metabolism are often implemented using multi-component molecular assemblies. Understanding how these molecular machines function is an emerging frontier in cell biology, which will begin to bridge the gap that exists between our knowledge of the structures of individual proteins and those of cellular organelles. As more networks of interacting proteins emerge from genomics and proteomics, the need for innovative methods to illuminate these potentially disordered complexes will amplify. A major focus of my laboratory is the structure determination of large multiprotein complexes by analysis of high resolution images of single molecules. In single particle electron microscopy, images containing large numbers of well-separated protein molecules are recorded using low-dose electron microscopy of frozen-hydrated samples. Individual molecules are computationally selected, sorted into distinct classes, and averaged 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, which is subsequently improved using specific refinement algorithms. Pyruvate dehydrogenase multienzyme complexes are among the largest protein assemblies within cells, and catalyze the synthesis of acetyl CoA from pyruvate, a key metabolic reaction. While NMR and X-ray crystallographic studies have provided atomic models for the individual protein components that constitute this complex, determination of the structure of the entire complex, as well as an understanding of the mechanism of active site coupling have remained elusive goals. Using high resolution electron microscopy, we have arrived at an atomic interpretation of an 11,000 kDa icosahedral complex containing 60 copies of 2-oxo-acid decarboxylase (E1 enzyme) and 60 copies of dihydrolipoyl acetyltransferase (E2 enzyme). The molecular model provides a unique glimpse of the architecture and inner workings of a fascinating cellular machine, showing how the complex uses a swinging arm mechanism to couple the active sites of the E1 and E2 enzymes which are located at a distance of approximately 100 ? from each other. We have also analyzed the 1,800 kDa icosahedron formed by the E2 core enzyme to obtain a 14 ? model that is in excellent agreement with the 4.5 ? structure determined by X-ray crystallography. We are using this model system to optimize methods required to accurately orient the molecules, to correct distortions introduced during image collection on the electron microscope, and to enhance the speed of data processing so that it will be possible to analyze the hundreds of thousands of molecular images that will be required to attain near-atomic resolution three-dimensional models of non-symmetrical molecules. Continued refinement of single particle methods to facilitate the analysis of large dynamic complexes may provide a powerful tool to investigate other important macromolecular complexes present in normal and malignant cells.

复杂的细胞过程，如信号转导、基因表达、运动和能量代谢，通常是通过多组分的分子组装来实现的。了解这些分子机器的功能是细胞生物学的一个新兴前沿，这将开始弥合我们对单个蛋白质的结构和细胞细胞器的结构之间存在的差距。随着基因组学和蛋白质组学出现更多相互作用的蛋白质网络，对阐明这些潜在的无序复合体的创新方法的需求将被放大。我的实验室的一个主要重点是通过分析单分子的高分辨率图像来确定大型多蛋白复合体的结构。在单粒子电子显微镜中，包含大量分离良好的蛋白质分子的图像是使用冷冻水化样品的低剂量电子显微镜记录的。通过计算选择单个分子，将其分成不同的类别，并将其平均在一起，以获得具有高信噪比的分子的不同视图。然后，平均的视图被彼此定向，并用于重建三维结构的模型，该模型随后使用特定的细化算法来改进。 丙酮酸脱氢酶多酶复合体是细胞内最大的蛋白质组合之一，催化丙酮酸合成乙酰辅酶A，这是一个关键的代谢反应。虽然核磁共振和X射线结晶学研究已经为组成这种复合体的单个蛋白质组分提供了原子模型，但确定整个复合体的结构以及了解活性位点耦合的机制仍然是一个难以实现的目标。利用高分辨电子显微镜，我们得到了一个11,000 kDa的二十面体络合物的原子解释，该络合物包含60个拷贝的2-氧基酸脱羧酶(E1酶)和60个拷贝的二氢硫辛基乙酰转移酶(E2酶)。分子模型提供了一个迷人的细胞机器的结构和内部工作的独特一瞥，展示了如何使用摆臂机制耦合位于大约100？彼此之间的距离。 我们还分析了由E2核心酶形成的1800 kDa二十面体，得到了14？这款机型与4.5版非常吻合？由X射线结晶学确定的结构。我们正在使用这个模型系统来优化精确定位分子所需的方法，纠正在电子显微镜上采集图像时引入的扭曲，并提高数据处理的速度，以便有可能分析达到近原子分辨率的非对称分子的三维模型所需的数十万分子图像。不断完善的单粒子方法有助于分析大的动态复合体，这可能为研究存在于正常细胞和恶性肿瘤细胞中的其他重要的大分子复合体提供一个强有力的工具。