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Updated: May 30, 2026

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy
Published on: March 3, 2023
Cryo-electron tomography of microtubules assembled in vitro from purified components
Frédéric M Coquelle1, Sophie Blestel, Claire Heichette
1CNRS, UMR 6026 Interactions Cellulaires et Moléculaires, IFR 140 Génomique Fonctionnelle Agronomie et Santé, Université de Rennes 1, Rennes, France.
Abstract:
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.
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