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

开发结构分析和电子显微镜以可视化单运动蛋白

• 批准号: 06558098
• 负责人: WAKABAYASHI Takeyuki
• 金额: $ 5.38万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06558098/
• 关键词: Muscle; Myosin; Actin; Tubulin; Kinesin; Electron microscopy; X-ray scattering; Site-directed mutagenesis; クライオ電子顕微鏡法; 対物レンズ; 傾斜像シリーズ; 画像解析; ミオシン; 筋収縮; アンデカゴールド

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.

我们的目标是根据蛋白质的原子结构来了解运动蛋白的分子机制。首先，我们用冷场发射枪启用了电子显微镜。特别是，我们开发了一个新的目标Lenz，可以通过它观察到高向图像，一种抗抗激素，是一种新的最低剂量系统。第二，我们开发了新的图像分析技术。第一个是电子层析成像，用于重建跨桥的三维图像在收缩肌肉中。第二个是使用全息图像重建技术对单运动蛋白的可视化。第三个是三维重建，而无需使用螺旋对称性以重建薄丝的三维结构。第四个是一种使用螺旋对称性的新的三维结构。三分之二，通过电子冷冻微观，我们分析了动力蛋白 - 细胞蛋白 - 细丝，细丝，天然细丝，肌动肌蛋白络合物，肌动蛋白络合物和肌动蛋白丝的三维结构，我们确定了肌动蛋白N-末端的位置。冷冻电子显微镜。

项目成果

Ishikawa, T.& Wakabayashi T.: "Calcium induced change in three-dimensional structure of thin filaments of rabbit skeletal muscle as revealed by cryo-electron microscopy" Biochem.Biophys.Res.Commun.203. 951-958 (1994)

石川，T.

Ishikawa,T.& Wakabayashi,T.: "Proposal of Alignment-Independent Classification of Electron Microscopic Images with Helical Symmetry and its Application to Reconstituted Thin Filaments of Skeletal Muscle" Ultramicroscopy. 57. 91-101 (1995)

石川，T.

Yamakawa, H., Seog, D.-H., Yoda, K., Yamasaki, M., & Wakabayashi, T.: "Uso1 protein is a dimer with two globular heads and a long coiled-coil tail" J.Struct.Biol.116. 356-365 (1996)

山川，H.，西奥格，D.-H.，尤达，K.，山崎，M.，

Meng, Y. et al.: "Detemination of the Protein Components of Native Thin Filaments Isolated from Natural actomyosin: Nebulin and a-Actinin Are Associated with Actin Filaments" J. Biochem.118. 422-427 (1995)

孟，Y.等人：“从天然肌动球蛋白中分离出的天然细丝的蛋白质成分的测定：星云蛋白和α-肌动蛋白与肌动蛋白丝相关”J.Biochem.118。

Saeki, K., Sutoh, K.& Wakabayashi, T.: "T., Tropomyosin-binding site(s) on the Dictyosterium Actin surface as identify by site-directed mutagenesis" Biochemistry. 35. 14465-14472 (1996)

佐伯，K.，须藤，K.