STRUCTURE OF TUBULIN IN AN UNCONSTRAINED GDP BOUND STATE

这个项目的最终目标是了解 微管蛋白与核苷酸水解有关，并导致动态 微管的性质。为此，我们将比较 两种不同构象的微管蛋白二聚体代表高和低 蛋白质的能态：直原丝中的构象 在这种情况下，微管蛋白-GDP分子被限制在“GTP样”状态 聚合物晶格和弯曲的环上的构象 微管蛋白-GDP处于松弛状态的原丝。 约束状态下微管蛋白的结构是近年来研究的热点。 在唐宁实验室获得的锌诱导的电子结晶学 床单。目前的项目将涉及获得该结构 玻璃化样品的冷冻电子显微镜观察微管蛋白环 基于单粒子方法的图像重建。立即采取的措施将是 是为了获得中分辨率(约15A)的投影 环形结构。对预测的解释将得到以下帮助 与片状约束微管的原子模型进行了比较。 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，使我们能够测试所提出的假设 核苷酸含量对微管蛋白二聚体结构的影响。 下一步将涉及提高分辨率的重建，如 数据收集和处理工作取得了进展。10岁时的重建 盎司将不仅考验我们整体形状的变化 二聚体，但是否有任何三级结构重排在 分子；哪些二级结构元素受影响最大 核苷酸状态；以及这些结构变化可能如何影响组装 微管蛋白的性质。

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.