Atomic Resolution Biological Electron Microscopy

我们的研究项目集中在使用高分辨率电子显微镜分析大分子组装体的三维结构。我们小组的成员致力于结构分析的不同但互补的方面。这些包括二维蛋白质晶体的电子晶体学研究，分析蛋白质复合物结构的“单粒子”方法，以及使用电子断层扫描确定大型亚细胞组装体的结构。我们的研究工作的一个重要组成部分是致力于开发和实施新的方法，样品制备，高通量数据采集和计算分析。 2000-2001年研究重点： ?我们完成了对细菌视紫红质结构变化的全面分析，使我们能够提出这种蛋白质光驱动质子运输的详细原子机制。除了新的生物学见解，我们的蛋白质开放构象的原子分辨率结构是最高分辨率（~ 3？）这是通过电子显微镜在任何膜蛋白上获得的。 ?我们已经成功地结晶细菌草酸转运，并已确定在6？分辨率这项工作提供了一个非常大的转运蛋白超家族的架构的第一个见解。 ?我们已经开发出了在生物电子显微镜中引入高水平自动化的方法。这项工作已经对我们实验室的数据收集速度产生了相当大的影响。NCI已经就这项技术提交了临时专利申请。 ?我们从B导出了11，000 kD丙酮酸脱氢酶复合物的原子模型。嗜热脂肪菌该模型是通过对接在四个单独的组成部分的酶，其结构已确定由X-射线晶体学到一个中等分辨率的电子显微镜模型的整个复杂的。结果是非常令人兴奋和意想不到的。 ?我们已经证明，CCD探测器现在可以完全取代照相胶片记录电子衍射图案的分辨率为2？，并有积极的兴趣继续改善检测器技术的高分辨率电子显微镜通过测试更大和更好的CCD相机的分子成像应用。 在未来的几年里，我们的计划是继续在NCI/NIH开发高分辨率电子显微镜的基础设施，并使用三种主要类型的方法来解决基本的生物学问题： ?近原子分辨率的膜蛋白电子晶体学，特别关注在生物膜中作为转运蛋白的蛋白质 ?分子电子显微镜通过分析“单粒子”图像来确定大分子复合物的结构，例如多蛋白质细胞机器和尺寸范围从200 kD到10，000 kD的复杂组件 ?电子断层扫描确定非常大的分子组装体、亚细胞器和细胞切片的三维结构 我们的实验室目前配备了一个300千伏场发射枪，和两个标准的120千伏电子显微镜。我们还有一个高端计算机器集群，其中包括几台DEC阿尔法、SGI和Linux机器，用于图像处理。

Our research program is focused on the analysis of the three-dimensional structures of macromolecular assemblies using high resolution electron microscopy. Members of our group work on different, yet, complementary aspects of structural analysis. These include electron crystallographic studies of two-dimensional protein crystals, "single particle" approaches to analyze the structures of protein complexes and determination of the structures of large subcellular assemblies using electron tomography. A significant fraction of our research effort is devoted to developing and implementing novel methods for specimen preparation, high throughput data acquisition and computational analysis. Research Highlights 2000-2001: ? We completed a comprehensive analysis of structural changes by bacteriorhodopsin that allowed us to present a detailed atomic mechanism for the light-driven transport of protons by this protein. Aside from the new biological insights, our atomic resolution structure for the open conformation of the protein is at the highest resolution (~ 3?) that has been obtained on any membrane protein by electron microscopy. ? We have successfully crystallized a bacterial oxalate transporter, and have determined a projection structure at 6 ? resolution. This work provides the first insights into the architecture of a very large superfamily of transporter proteins. ? We have developed methods to introduce a high level of automation in biological electron microscopy. This work has already had a considerable impact on the speed of data collection in our laboratory. NCI has filed a provisional patent application on this technology. ? We have derived an atomic model for the 11,000 kD pyruvate dehydrogenase complex from B. stearothermophilus. The model was obtained by docking in four individual components of the enzyme whose structures have been determined by X-ray crystallography into a medium resolution electron microscopic model of the entire complex. The results have been very exciting and unexpected. ? We have demonstrated that CCD detectors can now fully replace photographic film to record electron diffraction patterns to resolution of 2 ?, and have an active interest in continuing to improve detector technology for high resolution electron microscopes by testing bigger and better CCD cameras for molecular imaging applications. In the coming years, our plan is to continue to develop an infrastructure for high resolution electron microscopy at NCI/NIH and to address fundamental biological problems using three principal types of methodologies: ? Electron crystallography of membrane proteins, at near-atomic resolution with a particular focus on proteins that function as transporters in biological membranes ? Molecular electron microscopy to determine the architectures of large macromolecular complexes such as multi-protein cellular machines and complex assemblies with sizes ranging from 200 kD to 10, 000 kD by analysis of "single particle" images ? Electron tomography to determine the three-dimensional architectures of very large molecular assemblies, subcellular organelles and cellular sections Our laboratory is currently equipped with a 300 kV field emission gun, and two standard 120 kV electron microscopes. We also have a cluster of high end computing machines that include several DEC Alphas, SGIs, and Linux machines for image processing.