STRUCTURAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的长期目标是开发出 大分子组装的结构和功能研究 单粒子(非晶态)形态，使用低剂量电子显微镜 冰冻水合的样品。现有的成功的对准方法， 分类，并在校长和重建发展 调查员的实验室将向更高的分辨率扩展， 10A被设定为名义目标。与领先的 调查人员将提供供应技术领域的专业知识 用于研究的材料。 具体地说，将作出以下技术开发努力： (一)传递函数校正，并澄清 非弹性散射；(2)数据合并和重建的改进 方法--投影数据的合并类似于数据的合并 在电子结晶学中，除了取向也必须是 改进；(3)发展点扫描、数字读出和 提高低温实验成品率和数据效率的诊断 收集；(4)通过以下方法进一步探索修复 投影到凸集(POCS)，以消除丢失的数据锥 在随机圆锥重建中；(V)二维和三维图像的发展 建模，模拟电子显微镜的动作并预测 为提出的模型观察到的图像，例如核糖体RNA。这将是 允许对实验数据和理论数据进行比较。 建议的具体结构和功能研究侧重于 大肠杆菌和哺乳动物核糖体。这些研究承诺 解开蛋白质合成机制中的一些步骤，它们是 在理解某些抗体的作用方面具有核心重要性 细菌疾病和强效毒素的作用。T-RNA、因子和 抗体图谱将在三个维度上使用，以获取信息 在翻译过程中。将在中调查的其他系统 与法国实验室合作的是血蓝蛋白和人类α-2 巨球蛋白。

The long-term objective of this project 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. Existing successful methods of alignment, classification, and reconstruction developed in the principal investigator's laboratory will be extended toward higher resolution, with 10 A being set as a nominal goal. Several collaborations with leading investigators will provide expertise in technical areas of supply material for research. Specifically, the following technical development efforts will be made: (i) transfer function correction, and clarification of the role of inelastic scattering; (ii) refinement of data merging and reconstruction methods -- merging of projection data is analogous to the merging of data in electron crystallography, except that the orientation must also be refined; (iii) development of spot scanning, digital readout and diagnosis to improve the yield of cryo-experiments and efficiency of data collection; (iv) further exploration of restoration, by the method of projection onto convex sets (POCS), to eliminate the missing data cone in the random-conical reconstruction; (v) development of 2D and 3D image modeling, to simulate the action of the electron microscope and predict images observed for models proposed, e.g. for ribosomal RNA. This will allow comparison between experimental and theoretical data. The specific structural and functional studies proposed focus on the Escherichia coli and the mammalian ribosome. These studies promise to unravel some of the steps in the mechanism of protein synthesis which are of central importance in understanding the action of certain antibodies in bacterial diseases and the action of potent toxins. t-RNA, factor and antibody mapping will be used in three dimensions to obtain information on the translation process. Other systems that will be investigated in collaboration with a French laboratory are hemocyanin and human alpha-2 macroglobulin.