Structural basis of allostery and mechanical properties of F-actin

 描述（由申请人提供）：我们将采用多学科的实验方法，包括分子细胞生物学，生物化学，生物物理方法，计算分析和高分辨率冷冻电子显微镜（cryo-EM），以提供不同功能状态下肌动蛋白丝（F-肌动蛋白）的结构。在3 - 5 μ m的目标分辨率下，α-螺旋和β-链将被分辨，大的侧链将可见，从而允许多肽主链和许多侧链的准确放置。冷冻电子断层扫描将补充这些努力，提供地图，而不应用螺旋对称，允许可视化的原聚体的变化，在灯丝。我们将充分利用低温EM领域的最新技术进步，特别是直接探测器成像设备，相位板技术，基于最大似然的数据处理和强大的数据收集设备（Titan Krios）。因此，通过直接测定F-肌动蛋白原聚体在微丝内和与肌动蛋白结合蛋白（ABP）的结构和界面，并通过检查原聚体构象沿着肌动蛋白微丝的变化，我们将解决F-肌动蛋白功能的三个关键方面：（i）解释ABP与F-肌动蛋白结合亲和力的核苷酸依赖性;（ii）解释ABP与F-肌动蛋白结合的协同性;和（iii）定义F-肌动蛋白刚度调节的结构基础。

 DESCRIPTION (provided by applicant): We will employ a multidisciplinary experimental approach that includes molecular cell biology, biochemistry, biophysical approaches, computational analysis, and high-resolution cryo electron microscopy (cryo-EM) to provide structures of actin filaments (F-actin) in different functional states. At our target resolution of3-5Å, α- helices and β-strands will be resolved and large side chains will be visible allowing accurate placement of the polypeptide backbone and of many side chains. Cryo electron tomography will supplement these efforts by providing maps without the application of helical symmetry allowing visualization of protomer variability in the filament. We will take full advantage of recent technical advances in the cryo-EM field, specifically direct detector imaging devices, phase plate technology, maximum likelihood based data processing, and robust data collection equipment (Titan Krios). Thus, by direct determination of the structures and interfaces of F-actin protomers within the filament and with actin-binding proteins (ABPs) and by examining the variability of protomer conformations along actin filaments, we will address three key aspects of F-actin function: (i) to explain the nucleotide-dependence of ABP binding affinity to F-actin; (ii) to explain the cooperativity of ABP binding in F-actin; and (iii) to define the structural basis of F-actin stiffness regulation.