Coquelle Frédéric M, Blestel Sophie, Heichette Claire, Arnal Isabelle, Kervrann Charles, Chrétien Denis
CNRS, UMR 6026 Interactions Cellulaires et Moléculaires, IFR 140 Génomique Fonctionnelle Agronomie et Santé, Université de Rennes 1, Rennes, France.
Methods Mol Biol. 2011;777:193-208. doi: 10.1007/978-1-61779-252-6_14.
Cryo-electron tomography of vitrified specimens allows visualization of thin biological samples in three-dimensions. This method can be applied to study the interaction of proteins that show disorder and/or bind in a nonregular fashion to microtubules. Here, we describe the protocols we use to observe microtubules assembled in vitro in the presence of XMAP215, a large and flexible protein that binds to discrete sites on the microtubule lattice. Gold particles are added to the mix before vitrification to facilitate image acquisition in low-dose mode and their subsequent alignment before tomographic reconstruction. Three-dimensional reconstructions are performed using the IMOD software, processed with ImageJ and visualized in UCSF Chimera. Extraction of features of interest is performed using a patch-based algorithm (CryoSeg) developed in the laboratory. All the software used in this procedure is freely available or can be obtained on request, and run on most operating systems.
玻璃化标本的冷冻电子断层扫描能够实现对薄生物样本的三维可视化。该方法可用于研究表现出无序和/或以不规则方式与微管结合的蛋白质之间的相互作用。在此，我们描述了我们用于观察在XMAP215存在下体外组装的微管的实验方案，XMAP215是一种大的柔性蛋白质，可结合到微管晶格上的离散位点。在玻璃化之前将金颗粒添加到混合物中，以促进低剂量模式下的图像采集及其在断层重建之前的后续对齐。使用IMOD软件进行三维重建，用ImageJ进行处理，并在UCSF Chimera中进行可视化。使用实验室开发的基于补丁的算法（CryoSeg）提取感兴趣的特征。本程序中使用的所有软件均可免费获得或可根据要求获取，并且可在大多数操作系统上运行。
