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，通常是通过多组分的分子组装来实现的。了解这些多组分分子机器的功能是细胞生物学的一个新兴前沿，它将开始定义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信息鸿沟。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方法的需求将被放大。对于核磁共振或X射线结晶学技术都不适用的各种生物标本，高分辨率电子显微镜是迎接这一挑战的唯一准备。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，该模型随后使用细化算法来改进。 利用单分子显微镜，我们定义和解释了二十面体丙酮酸脱氢酶多酶复合体的结构，这是一个多步催化机器的典型例子，它耦合了丙酮酸氧化脱羧反应中三个组分酶(E1、E2和E3)的活性，在糖酵解和三羧酸循环的交界处生成乙酰辅酶A。合成了一个由60个E2酶和60个E1酶组成的11-丙二醛二十面体PDH复合体的三维模型。从电子冷冻显微镜获得的结构，具有先前确定的络合物各组分的原子坐标。对模型的分析为这种分子机器的设计和功能提供了许多新的见解。一个关键特征是，E1分子位于外围，其取向允许与内部E2酶连接的60个可移动的硫辛基结构域中的每一个从二十面体复合体内部访问多个E1活性部位。这种意想不到的结构为活性中心的偶联和催化速率的提高提供了一种高效的机制，我们认为这是通过络合物内核和外核之间受限环区中的硫辛基结构域的运动来实现的。我们目前正在提炼由60个E2酶和60个E3酶组成的第二个PDH复合体，以确定为什么在体内，60个E2分子的内部二十面体被次优的只有48个E1分子和6个E3分子结合形成外层蛋白质壳的结构基础。对E1E2和E2E3复合体的分析表明，尽管E3在天然复合体中的占有率很低，但硫辛基结构域可以延伸到足够远的距离，以介导生成乙酰辅酶A所需的E1和E2的活性部位偶联，并与E3相互作用，在硫辛基结构域中再生必要的二硫键。 我们还在积极工作，以确定导致突出显微图像的条件，开发方法来选择和准确地对齐用于三维重建的最佳分子图像，可靠地解释这些结构，并开发自动化程序来促进获得高质量的大分子复合体三维模型的过程。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，并评估了由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，结合网格线程蒙特卡罗方法，以增强可靠地识别单个组分的原子坐标与较大络合物的低分辨率图的最佳拟合的能力，这些图是使用单粒子电子显微镜确定的典型结构，以及4)用于计算分析分子图像的优化方法。后者涉及到精确定位分子的方法，纠正在电子显微镜上采集图像时引入的扭曲，以及提高数据处理的速度，以便有可能分析获得非对称分子的近原子分辨率三维模型所需的数十万分子图像。我们已经成功地设计了计算机程序，以使我们的图像分析程序与Biowulf-Lobos计算机集群接口。并行计算方法和基于Web的图形用户界面的开发已经使用43,000张额外的丙酮酸脱氢酶催化核心图像进行了测试，比我们之前的精炼过程快了两个数量级以上。E_2二十面体核心的分辨率由原来的14.5？比12岁还要好？已经开发了一个SQL数据库并将其链接到这些程序，以便于分析数十万个分子图像，这些图像将需要达到10？决议。不断改进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重要大分子复合体提供一个强有力的工具。

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.