Development of structural analysis and electron microscopy to visualize single motor protein

我们的目标是从蛋白质的原子结构上了解马达蛋白质的分子机制。首先，我们改进了一台带有冷场发射枪的电子显微镜。特别是，我们开发了一种新的物镜Lenz，通过它我们可以观察高倾斜图像、抗污染剂、新的最小剂量系统。第二，我们开发了新的图像分析技术。第一种是电子断层成像，用于重建收缩肌肉中跨桥的三维图像；第二种是利用全息图像重建技术对单个运动蛋白进行可视化。第三种是不利用螺旋对称性的三维重建，以重建细丝的三维结构。利用电子冷冻显微镜分析了肌动蛋白-微管蛋白复合体、细丝、天然细丝、肌动球蛋白复合体和肌动蛋白细丝的三维结构，最后利用蛋白质工程和冷冻电子显微镜确定了肌球蛋白N-末端在肌动蛋白-肌球蛋白刚性复合体中的位置。

Our goal is to understand the molecular mechanism of motor proteins on the basis of atomic structure of proteins.First, we inproved an electron microscope with a cold field emission gun. Especially, we developed a new objective lenz, by which we can observe high-tilted images, an anti-contaminator, a new minimum dose system.Second, we developed new image analysis techniques. The first one is electron tomography to reconstruct three-dimensional images of cross-bridge in contracting muscle.The second one is visualization of single motor protein using a holographic image reconstruction technique. The third one is three-dimensional reconstruction without using helical symmetry in order to reconstruct three-dimensional structure of thin filaments. The forth one is a new three-dimensional structure using helical symmetry.Third, by electron cryo-microscopy, we analyzed three-dimensional structure of kinesin-tubulin complexes, thin filaments, native thin filaments, actomyosin complexes, and actin filaments.Last, we identified the position of the myosin N-terminus in the actin-myosin rigor complexes using protein engineering and cryo-electron microscopy.