Structural Analysis of Macromolecular Complexes by High

复杂的细胞过程，如信号转导，基因表达，运动和能量代谢，往往是使用多组分分子组装。了解这些多组分分子机器如何发挥作用是细胞生物学的一个新兴前沿，这将开始定义我们对单个蛋白质结构和细胞器结构的知识之间存在的信息差距。随着基因组学和蛋白质组学中出现更多相互作用的蛋白质网络，对阐明这些潜在无序复合物的方法的需求将扩大。高分辨率电子显微镜是唯一的准备，以满足这一挑战的各种生物标本，既不适合核磁共振或X射线晶体学技术。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质复合物的结构。在单粒子电子显微镜中，使用冷冻水合样品的低剂量电子显微镜记录包含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，将其分类为不同的类别，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，平均视图相对于彼此定向，并用于重建三维结构的模型，该模型随后使用细化算法进行改进。 使用单分子显微镜，我们已经定义和解释的二十面体丙酮酸脱氢酶多酶复合物的结构，一个典型的例子，一个多步骤的催化机器耦合的活性的三个组成部分的酶（E1，E2和E3）在丙酮酸的氧化脱羧生成乙酰辅酶A的糖酵解和三羧酸循环的交界处。的三维模型为11 MDa，二十面体PDH复合物，由60 E2酶和60 E1酶，通过结合28？来自电子低温显微镜的结构，具有先前确定的复合物的各个组分的原子坐标。对该模型的分析为这种分子机器的设计和功能提供了一些新的见解。一个关键特征是E1分子位于外围，其取向允许60个移动的硫辛酰基结构域中的每一个连接到内部E2酶，以从二十面体复合物内部访问多个E1活性位点。这种意想不到的架构提供了一个高效的机制，活性位点耦合和催化速率的增强，我们建议是通过运动的硫辛酰域之间的复杂的内部和外部核心的限制性环形区域。我们目前正在改进由60个E2酶和60个E3酶组成的第二种PDH复合物，以确定为什么在体内60个E2分子的内部二十面体被仅约48个E1分子和6个E3分子次优地占据以通常结合形成外部蛋白质壳的结构基础。E1 E2和E2 E3复合物的分析表明，尽管E3在天然复合物中的占有率低，硫辛酰基结构域可以延伸到足够远，以介导产生乙酰辅酶A所需的E1和E2的活性位点偶联，并与E3相互作用，以再生硫辛酰基结构域中的必需二硫键。 我们还积极致力于确定导致出色的显微图像的条件，开发选择和准确对齐三维重建的最佳分子图像的方法，可靠地解释这些结构，并开发自动化程序以促进获得高质量大分子复合物三维模型的过程。为此，我们1）开发了在Tecnai系列电子显微镜上自动收集数据的算法，2）表征了4000 x 4000像素数字CCD相机的特性并评估了从CCD数字图像构建的三维分子模型的质量，3）开发了“核心加权”方法，与网格线程蒙特卡罗方法相结合，以增强可靠地将单个组分的原子坐标的最佳拟合识别到较大复合物的低分辨率图中的能力，所述较大复合物是使用单粒子电子显微镜确定的典型结构，以及4）分子图像计算分析的优化方法。后者涉及的方法，以准确地定向的分子，以纠正在电子显微镜上的图像收集过程中引入的失真，并提高数据处理的速度，使它将有可能分析数十万的分子图像，将需要获得近原子分辨率的非对称分子的三维模型。我们已经成功地设计了计算机程序，旨在接口我们的图像分析程序与Biowulf-Lobos计算机集群。并行计算方法和基于网络的图形用户界面的发展已经过测试，使用43,000个额外的图像的催化核心的丙酮酸脱氢酶，是超过两个数量级的速度比我们早期的细化程序。E2二十面体核心的分辨率已从14.5？比12更好吗？.一个SQL数据库已经开发出来，并连接到这些程序，以方便分析的数十万的分子图像，将需要达到超过10？分辨率单粒子方法的不断改进，以促进大的动态复合物的分析，可能会提供一个强大的工具，调查存在于正常和恶性细胞中的重要大分子复合物。

Complex cellular processes such as signal transduction, gene expression, motility and energy metabolism are often implemented using multi-component molecular assemblies. Understanding how these multi-component molecular machines function is an emerging frontier in cell biology, which will begin to define the information 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 methods to illuminate these potentially disordered complexes will amplify. High resolution electron microscopy is uniquely poised to meet this challenge for a variety of biological specimens that are amenable neither by NMR or X-ray crystallographic techniques. 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 refinement algorithms. Using single molecule microscopy, we have defined and interpreted the structure of an icosahedral pyruvate dehydrogenase multienzyme complex, a prototypical example of a multi-step catalytic machine which couples the activity of three component enzymes (E1, E2, and E3) in the oxidative decarboxylation of pyruvate to generate acetyl CoA at the junction of glycolysis and the tricarboxylic acid cycle. The three-dimensional model for a 11 MDa, icosahedral PDH complex, composed of 60 E2 enzymes and 60 E1 enzymes, was obtained by combining a 28 ? structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual components of the complex. Analysis of the model provides a number of novel insights into the design and function of this molecular machine. A key feature is that the E1 molecules are located on the periphery in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 enzyme to access multiple E1 active sites from inside the icosahedral complex. This unanticipated architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement, which we propose is achieved by the motion of the lipoyl domain in the restricted annular region between the inner and outer cores of the complex. We are currently refining a second PDH complex comprised of 60 E2 enzymes and 60 E3 enzymes to determine the structural basis of why in vivo the inner icosahedron of 60 E2 molecules is suboptimally occupied with only ~48 E1 molecules and 6 E3 molecules typically binding to form the outer protein shell. Analysis of the E1E2 and E2E3 complexes indicate that despite the low occupancy of E3 in the native complex, the lipoyl domains can extend far enough to both mediate active site coupling of E1 and E2 required for the generation of acetyl CoA, and to interact with E3 for the regeneration of an essential disulfide linkage in the lipoyl domain. We are also working actively to identify conditions that lead to outstanding microscopic images, to develop methods to select and accurately align the best molecular images for three-dimensional reconstructions, to reliably interpret these structures, and to develop automated procedures to facilitate the process of obtaining high quality three dimensional models of macromolecular complexes. To this end, we have 1) developed algorithms to collect data automatically on the Tecnai series of electron microscopes, 2) characterized the properties of a 4000 x 4000 pixel digital CCD camera and assessed the quality of the three-dimensional molecular models constructed from CCD digital images , 3) developed a "core-weighting" method, combined with a grid-threading Monte Carlo approach to enhance the ability to reliably identify the best fit of atomic coordinates of individual components into low resolution maps of larger complexes that are typical of structures determined with the use of single particle electron microscopy and 4) optimized methods for the computational analysis of molecular images. The latter involves methods 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. We have successfully designed computer programs designed to interface our image analysis programs with the Biowulf-Lobos computer cluster. The development of parallel computing methodology and a web-based graphical user interface has been tested using 43,000 additional images of the catalytic core of pyruvate dehydrogenase, and is over two orders of magnitude faster than our earlier refinement procedure. The resolution of the E2 icosahedral core has improved from 14.5 ? to better than 12 ? . A SQL database has been developed and linked to these programs to facilitate analysis of the hundreds of thousands of molecular images that will be required to reach better than 10 ? resolution. Continued refinement of single particle methods to facilitate the analysis of large dynamic complexes may provide a powerful tool to investigate important macromolecular complexes present in normal and malignant cells.