Three-dimensional image analysis of myosin head in function as captured by quick-freeze deep-etch replica electron microscopy.
Three-dimensional image analysis of myosin head in function as captured by quick-freeze deep-etch replica electron microscopy.
复制标题
通过快速冷冻深蚀刻复制品电子显微镜捕获的肌球蛋白头功能的三维图像分析。
DOI：
10.1007/978-1-4684-6039-1_5
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发表时间：
1998
影响因子：
--
通讯作者：
N. Baba
中科院分区：
文献类型：
--
作者：
E. Katayama;G. Ohmori;N. Baba
快速冷冻深蚀刻电子显微镜结合云母片技术提供了蛋白质分子在溶液中的高对比度、高时间和空间分辨率的图像，其三维结构得到了很好的保存。因此，获得单个功能分子的结构信息可能是非常有用的，例如在体外运动测试条件下的肌球蛋白交叉桥。用这种方法，我们实际上可以证明，在严格的条件下，重型肌球蛋白(HMM)的两个头大多是直的，并以大约45度的倾角结合到肌动蛋白细丝上，而在体外滑动条件下，它们只通过一个角度广泛的角度与肌动蛋白结合。我们还证明了在ATP或ADP/无机钒(Vi)存在下，自由HMM头部强烈扭曲，而在没有核苷酸的情况下，几乎是直的构型。为了研究单个交叉桥的更详细的结构，我们试图重建单个HMM分子的分子内亚域的三维结构。我们拍摄了一系列单个HMM-ADP/Vi粒子的倾斜图像，并成功地通过滤波反投影获得了其三维图像，即使在有限的倾斜角范围内也是如此。通过与没有核苷酸的亚片段-1(S1)的原子模型进行比较，我们发现了一些很大的结构差异，这可能部分是由于核苷酸结合导致的构象变化。
Quick-freeze deep-etch replica electron microscopy combined with mica-flake technique provides high contrast, high time- and spatial-resolution images of protein molecules in solution, whose three-dimensional structure is well preserved. Thus, it might be quite useful to obtain structural information of individual functioning molecules, such as myosin crossbridges under in vitro motility assay conditions. With that method, we could actually show that both heads of heavy meromyosin (HMM) crossbridges are mostly straight and bound to actin filaments with about 45 degree tilt-angle under rigor conditions, whereas they attached to actin through only one head with a wide variety of angles under in vitro sliding conditions. We also demonstrated that free HMM heads are strongly kinked in the presence of ATP or ADP/inorganic vanadate (Vi) in contrast to almost straight configuration in the absence of nucleotide. To examine more detailed structure of individual crossbridges, we tried to reconstruct the three-dimensional architecture of intramolecular subdomains of single HMM molecule. We took a series of tilted images of single HMM-ADP/Vi particle and successfully obtained its 3-D image by filtered back-projection, even with restricted range of tilt-angles. By comparison of the reconstruction with the atomic model of subfragment-1 (S1) without nucleotide, we found some great structural difference, which partly might be attributable to the conformational change by nucleotide binding.
DOI：
10.1073/pnas.83.17.6272
发表时间：
1986-09-01
影响因子：
11.1
作者：
KRON, SJ;SPUDICH, JA
通讯作者：
SPUDICH, JA