STRUCTURAL ANALYSIS OF MACROMOLECULAR COMPLEX

细胞生物学中一个突出的挑战是在原子分辨率下定义发生在生物膜上的分子事件，如将细胞外信号转换为细胞内反应、能量转换以及小分子和离子的产生或运输。对这些过程的充分理解将需要捕获在其基态和活化态的膜蛋白的原子分辨结构，以及在细胞膜上成核的多蛋白质复合体的原子视图。我的实验室的一个主要重点是通过分析单个粒子的高分辨率图像来确定如此大的多蛋白组件的结构。在这种方法中，使用非常低的电子剂量记录下大量的单个蛋白质分子的图像，将其分类为不同的类别，然后添加到一起，以获得具有高信噪比的分子的不同视图。然后，将平均视图彼此定向，并用于重建三维结构的模型。这一领域的最新进展表明，这种没有晶体的结晶学方法可能具有巨大的潜力，可以探测与生物相关的络合物，这些络合物太大，无法用核磁共振方法进行分析，或者不容易在电子显微镜或X射线结晶学研究所需的二维或三维阵列中结晶。丙酮酸脱氢酶的催化核心是单粒子法精制的优良模型体系。这种酶的60个拷贝，E2CD，组装成一个1800 kDa的二十面体复合体，很容易纯化，并在电子显微镜上显示出许多不同的取向。我们正在优化方法，以准确地定位单分子，并纠正在电子显微镜上采集图像时引入的扭曲。目前，对4500个单独的分子图像的处理已经产生了分辨率为14+的三维模型。该结构的预测包络与最近确定的X射线结构符合得很好。当催化核心与辅酶E1或E3络合时，分析得到了分辨率为35+的三维模型。即使在中等分辨率下，对这种络合物的研究也是有用的，因为通过X射线结晶学解析的单个组分的三维结构可以对接到由电子显微镜确定的密度图中。这些方法的改进也将有助于人类P-糖蛋白的分析，以及目前正在实验室进行的其他膜蛋白的相关结构项目。

An outstanding challenge in cell biology is to define at atomic resolution, the molecular events that occur at biological membranes such as the transduction of extracellular signals into intracellular responses, energy conversion, and the generation or transport of small molecules and ions. A full understanding of these processes will require atomic resolution structures of membrane proteins trapped in their basal and activated states, as well as atomic views of multi-protein complexes that nucleate at cellular membranes. A major focus of my laboratory is the structure determination of such large multiprotein assemblies by analysis of high resolution images of single particles. In this method, large numbers of images of individual protein molecules are recorded using very low electron doses, sorted into distinct classes, and then added 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. The pace of recent progress in this field suggests that this approach of crystallography without crystals may have great potential to probe biologically-relevant complexes that are too large to be analyzed by NMR methods or that do not crystallize easily in the two- or three-dimensional arrays required for electron microscopic or X-ray crystallographic studies. The catalytic core of the pyruvate dehydrogenase is an excellent model system for refinement of single particle methods. Sixty copies of this enzyme, E2CD, assemble into a 1,800 kDa icosahedral complex that is readily purified and that shows a number of distinct orientations on electron micrographs. We are optimizing methods to accurately orient the single molecules and to correct distortions introduced during image collection on the electron microscope. Currently, processing of 4500 individual molecular images has led to a three-dimensional model that has a resolution of 14 +. The predicted envelope of this structure is in excellent agreement with the recently determined X-ray structure. Analysis of the catalytic core when it is complexed to the accessory enzymes E1 or E3 has yielded a three-dimensional model that has a resolution of 35 +. The study of such complexes is useful even at modest resolution since three dimensional structures of the individual components solved by X-ray crystallography can be docked into density maps determined by electron microscopy. Refinement of these methods will also be useful for the analysis of human P-glycoprotein and for related structural projects of other membrane proteins currently underway in the laboratory.