Cryo Electron Microscopy of Acto-S1 ATPase Intermediates

描述（由申请人提供）：现在有强有力的（如果不是压倒性的）证据表明，基于肌球蛋白的运动主要是由于运动域附着在肌动蛋白上时杠杆臂角度的变化，即所谓的倾斜杠杆机制。现在需要的是更详细地了解这一机制，特别是不同生化和结构状态之间的相关性，不同肌球蛋白类型中运动机制的细节，以及单独的倾斜机制是否提供了运动的完整解释。我们工作的一个主要方面将是使用时间分辨电子冷冻显微镜，它允许标本在非常接近本地的条件下进行检查，并具有极好的时间分辨。我们将使用新的图像处理方法和自动化电子显微镜来获得高分辨率图像所需的大量数据。我们的目标是实现< 1nm的分辨率，这使得分子内部的α螺旋得以揭示。如果能做到这一点，就有令人兴奋的前景可以看到单个元素，其中有几个是α螺旋，以及它们如何在不同的生化状态下变化以产生运动。我们的大部分工作将使用肌凝蛋白v。它与肌动蛋白的“弱”结合状态比肌凝蛋白II具有更高的亲和力，从而可以研究力量中风前的构象。此外，大尺寸和不对称性（6个钙调素在调节区域）使肌球蛋白V特别适合于电子显微镜。然而，我们也将探索使用非肌肉形式的肌球蛋白II (NMIIA和NMIIB)，它们在肌动蛋白存在下的产物释放速度非常慢。我们的工作与许多涉及肌凝蛋白的疾病直接相关。肌球蛋白V已被确定为导致小鼠“稀释突变”的基因，这种突变会导致色素向毛囊的运输缺陷，也会导致发育和神经系统疾病。非肌球蛋白II在血小板活化和凝血调节中起关键作用。

DESCRIPTION (provided by applicant): There is now strong if not overwhelming evidence that myosin-based movement results largely from a change in lever arm angle while the motor domain is attached to actin, in what is called the tilting lever mechanism. What is now required is to understand this mechanism in considerably greater detail, especially the correlation between different biochemical and structural states, details of the movement mechanisms in different myosin types, and whether the tilting mechanism alone provides a complete explanation of movement. A major aspect of our work will be to use time-resolved electron cryo-microscopy, which allows specimens to be examined under conditions very close to native and with excellent time resolution. We will use new image processing methods and automated electron microscopy to obtain the very large amounts of data that are necessary for high resolution images. Our goal will be to achieve <1 nm resolution, which allows internal alpha-helices in molecules to be revealed. If this can be done, there is the exciting prospect of seeing the individual elements, several of which are alpha helices, and how they change in different biochemical states to produce movement. Most of our work will use myosin V. Its 'weakly' bound states with actin have a much higher affinity than those of myosin II, allowing study of the pre-power stroke conformation. Moreover, the large size and asymmetry (6 calmodulins in the regulatory domain) make myosin V especially suitable for electron microscopy. However, we will also explore the use of non-muscle forms of myosin II (NMIIA and NMIIB) that have very slow rates of product release in the presence of actin. Our work is directly relevant to a number of diseases involving myosins. Myosin V has been identified as being the gene responsible for the "dilute mutation" in mice, which has defective transport of pigment to hair follicles, and is also responsible for developmental and neurological disorders. Non muscle myosin II plays key roles in platelet activation and the regulation of blood clotting.