CRYO ELECTRON MICROSCOPY OF ACTO-S1 ATPASE INTERMEDIATES

肌肉收缩的分子基础被认为是 肌球蛋白头附着肌动蛋白时的构型变化。这样的一个 变化可以是头部本身的构象变化，也可以是 这可能是肌动蛋白结合角度的改变。能量 因为众所周知，力量的产生来自于三磷酸腺苷的水解，但 “斜跨桥”假说仍然具有吸引力，它仍然存在 未经证实。此外，最近的一些数据表明，这种配置 在ATP和ADP状态下，附着的肌球蛋白头的数目可能是相似的。 这增加了替代力量产生的建议的份量 应该考虑各种机制。 检验倾斜桥假说的主要问题是 建立所谓的弱结合ACTO的结构是困难的。 在三磷酸腺苷存在时出现的肌球蛋白状态。相当一部分是 已知ADP中出现的强束缚态的结构 或者没有核苷酸。然而，弱结合的ATP态一直难以捉摸 因为它们在低蛋白质浓度下解离 肌球蛋白的中等分辨率(约3 nm)显微镜观察 头部(S1)复合体。我们最近发现了稳定弱势群体的条件 并一直在用电子冷冻方法研究它们的结构。 显微镜下水合的标本。已经获得的显微照片显示 稳态ATP水解过程中附着的S1构型的变化； 因此，这些数据与倾斜桥假说是一致的，但 不要证明这一点。 现在我们希望将这项工作扩展到研究运动学的结构。 定义了ATPase循环中的弱态。如果不同的动力学状态可以 被证明具有本质上不同的结构，它将提供强大的 支持倾斜交叉桥假说。这项工作将是 由允许时间解析的冻结的新方法促进 电子显微镜标本将在毫秒尺度上进行。 这项工作的主要目标是获得 适合的生物化学定义的弱连接肌球蛋白状态 用于图像处理方法。远距离的目标是获得低点 弱连接交叉桥态的分辨结构 与肌动蛋白和肌球蛋白的高分辨率晶体结构一起使用 补充肌动球蛋白的僵硬结构，最近 获得。我们相信，这种方法提供了很好的解决前景 最重要的问题是什么？ 肌肉功能已有二十多年了。

The molecular basis of muscle contraction is thought to be a configurational change in the myosin head while attached to actin. Such a change could either be a conformational change within the head itself, or it may be an alteration in the binding angle made with actin. The energy for force production Is known to come from ATP hydrolysis, but while the "tilting cross-bridge" hypothesis remains attractive, It is still unproven. Moreover, some recent data has suggested that the configuration of attached myosin heads may be similar in both the ATP and ADP states. This has added weight to suggestions that alternative force generation mechanisms should be considered. The main problem in testing the tilting bridge hypothesis has been the difficulty in establishing the structures of so-called weakly bound acto- myosin states that occur in the presence of ATP. A considerable amount is known about the structure of the strongly bound state that occurs in ADP or no nucleotide. However, weakly bound ATP states have been elusive because they dissociate at the low protein concentrations required for moderate resolution (approximately 3 nm) microscopy of the acto-myosin head (S1) complex. We recently found conditions that stabilize the weakly bound states and have been studying their structures by electron cryo- microscopy hydrated specimens. The micrographs already obtained show a variety of attached S1 configurations during steady- state ATP hydrolysis; such data are therefore compatible with the tilting bridge hypothesis but do not prove it. We now wish to extend this work to the study the structures of kinetically defined weak states in the ATPase cycle. If different kinetic states can be shown to have substantially different structures it will provide strong support for the tilting crossbridge hypothesis. This work will be facilitated by new methodology allowing time-resolved freezing of the electron microscope specimens to be carried out on the millisecond scale. The primary objective of this work is to obtain micrographs of biochemically defined weakly attached actomyosin states that are suitable for image processing methods. The long range goal is to obtain low resolution structures of weakly attached crossbridge states that can be used with the high resolutions crystal structures of actin and myosin to complement the actomyosin rigor structures that have been recently obtained. We believe this approach offers excellent prospects to resolve what has been arguably the single most important problem in understanding muscle function for more than two decades.