Structural Analysis of Macromolecular Complexes by High

细胞包含数以千计的多分子复合体，它们像微型工厂机器一样协同工作。对这些分子装置的结构和功能的详细了解是细胞生物学中非常感兴趣的问题。我们的研究工作集中在单个粒子的电子冷冻显微镜分析上，这是确定复杂细胞组件三维结构的强大方法。我们定义并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过程中三种酶(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。通过将来自电子冷冻显微镜的28结构与先前确定的复合体单个成分的原子坐标相结合，获得了11丙二醛二十面体PDH复合体的三维模型，该复合体由60个E2酶和60个E1酶组成。EMBO J.21,5587,2002)。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。我们刚刚完成了第二个PDH复合体的精制，该复合体由60个E2酶和60个E3酶组成，以确定该酶复合体催化反应的最终再生阶段的结构基础。我们的三维重建表明，与上面描述的E1E2复合体类似，E3同源二聚体的内核和外壳之间存在75的环状间隙。通过用10、20、40或60个E1四聚体或用10、20、40或60个E3同源二聚体装饰E2核心形成的部分占据复合体的图像分析也表明，E2和E1或E2和E3的密度分别为75-95。因此，发生在蛋白质外壳的酶之间的相互作用并不负责维持复合体的大小。相反，连接E2催化域和E2外围亚单位结合域的E2内部连接子必须是相当坚硬的径向辐条，为E1和E3分子的组织提供支架。E3定位于稍微靠近核心的位置，这表明摆动的硫辛基结构域可以有效地访问所有三种酶的活性部位，而不会离开环状空间。这一结构进一步证明了在乙酰辅酶A的产生和随后的酶再生步骤中活性位点偶联的高效机制，这是启动新的乙酰辅酶A产生周期所必需的。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，我们开发了自动数据收集的算法，将X射线结构自动拟合到CRO电子显微镜得出的密度图中，并优化了分子图像的计算分析方法。

Cells contain thousands of multimolecular complexes which work together much like miniature factory machines. A detailed understanding of the structure and function of these molecular devices is a problem of great interest in cell biology. Our research efforts focus on electron cryo-microscopic analysis of single particles, a powerful method to determine the three-dimensional architectures of complex cellular assemblies. 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 (Milne et al. EMBO J. 21, 5587, 2002). 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 have just completed refinement of a second PDH complex comprised of 60 E2 enzymes and 60 E3 enzymes to determine the structural basis of the final regeneration phase of the reaction catalyzed by this enzyme complex. Our three-dimensional reconstruction this complex indicates that, similar to the E1E2 complex described above, an annular gap of 75 exists between the inner core and the outer shell of E3 homodimers. Image analysis of partial occupancy complexes, formed by decorating the E2 core with 10, 20, 40 or 60 E1 tetramers or with 10, 20, 40 or 60 E3 homodimers, also indicates a 75-95 separation of the E2 and E1 or E2 and E3 densities. Thus, interactions occurring between the enzymes of the outer protein shell are not responsible for maintenance of the size of the complex. Rather, the E2 inner linkers that connect the E2 catalytic domains to the E2 peripheral-subunit binding domains must be fairly rigid radial spokes that provide a scaffold for an organization of E1 and E3 molecules. E3 localizes slightly closer to the core, suggesting that the swinging lipoyl domain can effectively access the active sites of all three enzymes, without leaving the annular space. This architecture provides further evidence of the highly efficient mechanism for active site coupling during both the production of acetyl CoA and the subsequent enzyme regeneration step that is required to initiate a new cycle of acetyl CoA production. 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 developed algorithms for automated data collection, automated fitting of X-ray structures into density maps derived by cro electron microscopy, and optimized methods for the computational analysis of molecular images.