Cryo-electron Tomography of Microtubules Assembled In Vitro from Purified Components
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.
Key wordsMicrotubule Tubulin Microtubule-associated proteins XMAP215 Cryo-electron tomography Fiducial markers Three-dimensional reconstruction Segmentation
This work was supported by grants from the French National Agency for Research (ANR PCV06_142769 and PCV07_190830) and from the Federative Research Institute of Rennes IFR140 Functional genomics, Agronomy and Health.
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