STRUCTURE OF TUBULIN IN AN UNCONSTRAINED GDP BOUND STATE

本项目的最终目标是了解 微管蛋白与核苷酸水解有关， 微管的性质为此，我们将比较 微管蛋白二聚体在两个不同的构象代表高和低 蛋白质的能量状态：直原丝的构象 其中微管蛋白-GDP分子被限制在“GTP样”状态， 聚合物晶格，以及弯曲环中的构象 微管蛋白-GDP处于松弛状态。 微管蛋白在约束状态下的结构最近已经被 在唐宁的实验室通过锌诱导的电子晶体学获得 薄膜.本项目将涉及获得结构 微管蛋白环的冷冻电子显微镜玻璃化样品， 使用单粒子方法进行图像重建。立即采取措施， 获得中等分辨率（约15 A）的投影， 环状结构对预测的解释将有助于 与限制性微管蛋白的原子模型进行比较。 微管蛋白的两种构象之间的一些一般差异可能 在这个阶段已经获得，使我们能够测试提出的假设 核苷酸含量对微管蛋白二聚体结构的影响。 接下来的步骤将涉及增加分辨率的重建， 数据收集和处理工作正在进行。10岁时重建 昂格玛将考验我们的不仅是整体形态的变化， 二聚体，但是否有任何三级结构重排， 分子;哪些二级结构元素最受 核苷酸状态;以及这些结构变化如何影响组装 tubulin的特性

This project has its final goal to understand the structural changes in tubulin related to nucleotide hydrolysis and resulting in the dynamic nature of microtubules. To this aim, we will compare the structures of the tubulin dimer in two different conformation representing the high and low energy states of the protein: the conformation in a straight protofilament where the tubulin-GDP molecule is constrained in a "GTP-like" state by the polymer lattice, and the conformation in rings where are curved protofilaments where tubulin-GDP is in a relaxed state. The structure of tubulin in the constrained state has been recently obtained in Downing's lab by electron crystallography of zinc-induced sheets. The present project will be concerned with obtaining the structure of the tubulin rings by cryo-electron microscopy of vitrified samples and image reconstruction using single particle methods. An immediate step will be to obtain a medium resolution (approximately 15 A) projection of the ring structure. The interpretation of the projection will be helped by comparison with the atomic model of the constrained tubulin in sheets. Some general differences between the two conformations of tubulin could already be gained at this stage, allowing us to test proposed hypothesis on the effect of nucleotide content on the structure of the tubulin dimer. The next steps will involves reconstructions of increasing resolution as the data collection and processing progresses. A reconstruction at 10 Angstroms will test us not only the overall change in the shape of the dimer, but whether there are any tertiary structure rearrangements in the molecule; which secondary structure elements are most affected by the nucleotide state; and how these structural changes may affect the assembly properties of tubulin.