Macromolecular Complex Structure /High Res. Electron Mic

复杂的细胞过程，如信号转导、基因表达、运动和能量代谢，通常是通过多组分分子组装来实现的。了解这些分子机器如何运作是细胞生物学的一个新兴前沿，它将开始弥合我们对单个蛋白质结构和细胞器结构之间存在的知识差距。随着更多相互作用的蛋白质网络从基因组学和蛋白质组学中出现，对创新方法的需求将扩大，以阐明这些潜在的无序复合物。我的实验室的一个主要焦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物的结构。在单粒子电子显微镜中，使用低剂量冷冻水合样品的电子显微镜记录含有大量分离良好的蛋白质分子的图像。通过计算选择单个分子，将其分为不同的类别，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。然后将平均视图相对于彼此定向，并用于重建三维结构模型，随后使用特定的细化算法对其进行改进。

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.