STRUCTURAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的长期目标是发展方法

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.