title: Cryo-electron tomography of microtubules assembled in vitro from purified components.
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.
authors.affiliation: CNRS, UMR 6026 Interactions Cellulaires et Moléculaires, IFR 140 Génomique Fonctionnelle Agronomie et Santé, Université de Rennes 1, Rennes, France.
authors.forename: Frédéric M
authors.initials: FM
authors.lastname: Coquelle
authors.forename: Sophie
authors.initials: S
authors.lastname: Blestel
authors.forename: Claire
authors.initials: C
authors.lastname: Heichette
authors.forename: Isabelle
authors.initials: I
authors.lastname: Arnal
authors.forename: Charles
authors.initials: C
authors.lastname: Kervrann
authors.forename: Denis
authors.initials: D
authors.lastname: Chrétien
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000648:ultrastructure; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure*; D014404:Tubulin / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D008869:Microtubule-Associated Proteins; D014404:Tubulin