Cryo Electron Microscopy of Acto-S1 ATPase Intermediates

Acto-S1 ATPase 中间体的冷冻电子显微镜

• 批准号: 7265091
• 负责人: HOWARD D. WHITE
• 金额: $ 34.04万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7265091
• 关键词: ATP phosphohydrolase; Accounting; Actins; Address; Affinity; Binding; Biochemical; Blood Clot; Blood coagulation; Calmodulin; Complex; Condition; Coupled; Cryoelectron Microscopy; Data; Development; Diffusion; Disease; Electron Microscopy; Electrons; Elements; Freezing; Genes; Goals; Hair follicle structure; Head; Helix (Snails); Image; Individual; Kinetics; Lead; Life; Methods; Microscopy; Molecular Conformation; Motor; Movement; Mus; Muscle; Mutation; Myosin ATPase; Myosin Type II; Myosin Type V; Negative Staining; Nucleotides; Numbers; Pigments; Platelet Activation; Play; Power stroke; Pre-study; Rate; Reaction; Regulation; Research Personnel; Resolution; Role; Route; Solutions; Specimen; Structure; Takeda brand of pioglitazone hydrochloride; Time; Tryptophan; Upper arm; Walkers; Walking; Work; alpha helix; base; cell motility; design; ear helix; image processing; improved; nervous system disorder; particle; programs; research study; size; time use

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.

描述(申请人提供)：现在有强有力的证据(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杠杆臂角度的变化，而运动域附着在肌动蛋白上，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，不同类型肌球蛋白运动机制的细节，以及倾斜机制是否单独提供了对运动的完整解释。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，它允许在非常接近自然条件下以极好的时间分辨率对标本进行检查。我们将使用新的图像处理方法和自动电子显微镜来获得高分辨率图像所需的大量数据。我们的目标将是达到&lt；1纳米的分辨率，这使得分子中的内部阿尔法螺旋得以揭示。如果能够做到这一点，就有可能看到单个元素，其中几个是阿尔法螺旋，以及它们如何在不同的生化状态下改变以产生运动的令人兴奋的前景。我们的大部分工作将使用肌球蛋白V，它与肌动蛋白的弱结合状态与肌球蛋白II的亲和力比肌球蛋白II高得多，从而可以研究强效前的中风构象。此外，肌球蛋白V的大小和不对称性(调节域中有6个钙调蛋白)使其特别适合于电子显微镜检查。然而，我们也将探索非肌肉形式的肌球蛋白II(NMIIA和NMIIB)的使用，它们在肌动蛋白存在的情况下具有非常缓慢的产物释放速度。我们的工作与许多涉及肌球蛋白的疾病直接相关。肌球蛋白V已被确定为导致小鼠“稀释性突变”的基因，这种突变具有色素到毛囊的运输缺陷，也是发育和神经疾病的罪魁祸首。非肌型肌球蛋白II在血小板活化和凝血调节中起关键作用。