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。 研究重点（2001-2002年）： (a)我们已经成功地结晶细菌草酸转运，并已确定在6？分辨率，和三维结构在6.5？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一瞥。 (b)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。 (c)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。 (d)我们正在开发的工具进行三维显微镜的整个细胞和部分使用新兴的方法在电子断层扫描。从细菌成像到整个脑组织的各种项目正在进行中。 (e)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？，并记录从“单个颗粒”到中等分辨率（~ 10？）的结构测定图像。

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 (2001-2002): (a) We have successfully crystallized a bacterial oxalate transporter, and have determined a projection structure at 6 ? resolution, and three-dimensional structure at 6.5 ? resolution. This work provides the first glimpse into the architecture of a very large superfamily of transporter proteins. (b) 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. (c) 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. (d) We are developing tools to carry out three-dimensional microscopy of whole cells and sections using newly emerging methods in electron tomography. A variety of projects ranging from imaging bacteria to whole brain tissue are underway. (e) We have demonstrated that CCD detectors can now fully replace photographic film to record electron diffraction patterns to resolution of 2 ?, and record images for structure determination from "single particles" to medium resolution (~ 10 ?).