STRUCTURAL ANALYSIS OF MACROMOLECULAR ASSEMBLY

这项研究计划的长期目标是开发方法 大分子组装体的结构和功能研究 单粒子(非晶态)形态，使用低剂量电子显微镜 冰冻的水合样本。最近发布的25埃3D 冷冻图谱是核糖体最成功的应用之一 到目前为止，这些方法中。随后，我们能够使用3D冷冻EM 差异作图定位翻译核糖体中的tRNA。这个 拟议的研究计划寻求抓住3D的新机遇 差值映射。具体地说，这项建议侧重于研究 伸长周期、亚基-亚基关联和 原核糖体中的新生多肽。我们也将开始 使用结构和功能探针以及3D冷冻-EM差异 酵母核糖体研究中的作图。 与这些调查携手，我们将继续努力 以提高分辨率。为此，我们将开发一种点扫描数据 收集程序，简化颗粒选择和处理以 使收集的粒子数量增加，并努力实现 提高了对生物颗粒成像的理解 冰。我们还将实施时间分辨技术来研究瞬变 TRNA的状态、因子结合和可能的构象变化 在伸长周期中的核糖体。

The long-term objective of this program of research is to develop methods of structural and functional studies for macromolecular assemblies in single-particle (noncrystalline) form, using low-dose electron microscopy of frozen hydrated samples. The recent publication of a 25 angstrom 3D cryo map of the ribosome has been one of the most successful application of these methods to date. Subsequently we were able to use 3D cryo EM difference mapping to locate tRNA in a translating ribosome. The proposed program of research seeks to seize the new opportunities of 3D difference mapping. Specifically, this proposal focuses on the study of the elongation cycle, subunit-subunit association and the path of the nascent polypeptide in the prokaryotic ribosome. We will also begin to use structural and functional probes along with 3D cryo-EM difference mapping in the study of the yeast ribosome. Hand in hand with these investigations, we will continue to make efforts to improve the resolution. To this end we will develop a spot scan data collection procedure, streamline particle selection and processing to enable collection of increased numbers of particles, and work towards an improved understanding of image formation for biological particles in ice. We will also implement time-resolved techniques to study transient states of tRNA, factor binding, and possible conformational changes of the ribosome during the elongation cycle.