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

我们的目标是在蛋白质原子结构的基础上了解运动蛋白的分子机制。首先，我们用冷场发射枪改进了电子显微镜。特别是，我们开发了一种新的物镜透镜，通过它可以观察高倾斜的图像，一个抗污染，一个新的最小剂量系统。其次，我们开发了新的图像分析技术。第一种方法是利用电子断层扫描重建收缩肌的三维交叉桥图像。二是利用全息图像重建技术实现单个马达蛋白的可视化。第三种是不使用螺旋对称的三维重建，重建细丝的三维结构。第四种是利用螺旋对称的新型三维结构。第三，通过电子冷冻显微镜，我们分析了运动蛋白-微管蛋白复合物、细丝、天然细丝、肌动球蛋白复合物和肌动蛋白细丝的三维结构。最后，我们利用蛋白质工程技术和冷冻电镜技术确定了肌动蛋白-肌凝蛋白复合体中肌凝蛋白n端的位置。

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.