Heavy Atom Electron Microscopy

描述（由申请人提供）：我们提出了一种新的策略， 单个生物颗粒的电子显微镜（EM）图像， 多个重原子团簇，严格固定在表面的特定位置 粒子，作为对齐标记。重原子团， 单独可见，并可以在EM图像中精确定位，将允许 精确确定粒子位置、均匀性和取向。的 该方法应能快速测定大多数生物颗粒的结构 在它们的天然、非结晶状态和生理条件下， 污染物如变形或亚化学计量颗粒的存在， 或具有可选择构象的颗粒。 我们这个项目的目标是证明该方法的可行性， 来创造通用的试剂和计算工具。在 前期工作，我们已经开发了计算机软件来模拟该方法， 我们已经建立了与蛋白质结合的四个簇的要求， 兴趣，我们估计了集群的固定化程度 为了进行高分辨率分析，我们准备了单链 针对合适的模型蛋白的抗体Fv片段（scFv），和 用大的金簇衍生化scFv，我们已经确定 保持高分辨率对星系团成像的条件 信息. 本项目的具体目标是：（1）优化scFv-簇 共轭这将需要scFv的定点诱变， 改变簇的化学性质以实现簇的刚性固定 需要高分辨率分析。(2)应用于蛋白质模型 已知结构。将所提出的方法的结果与 通过X射线晶体学获得的相同蛋白质将提供以下测试： 该方法和显示的分辨率，可以得到。(3)建设 修饰的scFv文库，以及计算工具，用于通过 生物医学研究界。

DESCRIPTION (provided by applicant): We propose a novel strategy to align electron microscope (EM) images of single biological particles that employs multiple heavy atom clusters, rigidly fixed to specific locations on the particles, as alignment markers. The heavy atom clusters, which are individually visible and can be precisely localized in EM images, will allow accurate determination of particle position, homogeneity, and orientation. The method should enable rapid structure determination of most biological particles in their native, uncrystalline state and under physiologic conditions, even in the presence of contaminants such as deformed or substoichiometric particles, or particles in alternative conformations. Our goals for this project are to demonstrate the feasibility of the method and to create the reagents and computational tools needed for general use. In preliminary work, we have developed computer software to simulate the method, we have established a requirement for four clusters bound to the protein of interest, we have estimated the degree of immobilization of the cluster necessary for high resolution analysis, we have prepared a single chain antibody Fv fragment (scFv) directed against a suitable model protein and derivatized the scFv with a large gold cluster, and we have determined conditions for imaging the cluster with the retention of high resolution information. Specific aims for the project period are: (1) Optimization of the scFv-cluster conjugate. This will entail site-directed mutagenesis of the scFv combined with variation of the cluster chemistry to achieve the rigid fixation of the cluster required for high resolution analysis. (2) Application to a model protein of known structure. Comparison of results from the proposed method with those obtained for the same protein by Xray crystallography will provide a test of the method and show the resolution that can be obtained. (3) Construction of a modified scFv library, as well as computational tools, for use of the method by the biomedical research community.