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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.
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Contributor : Charles Kervrann Connect in order to contact the contributor
Submitted on : Tuesday, December 13, 2011 - 2:51:56 PM
Last modification on : Wednesday, March 30, 2022 - 2:34:51 PM



Frédéric M. Coquelle, Sophie Blestel, Claire Heichette, Isabelle Arnal, Charles Kervrann, et al.. Cryo-Electron Tomography of microtubules assembled in vitro from purified components.. Methods in Molecular Biology, 2011, Microtubule Dynamics: Methods and Protocols, 777, pp.193-208. ⟨10.1007/978-1-61779-252-6_14⟩. ⟨hal-00651384⟩



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