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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Acknowledgments
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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Coquelle, F.M., Blestel, S., Heichette, C., Arnal, I., Kervrann, C., Chrétien, D. (2011). Cryo-electron Tomography of Microtubules Assembled In Vitro from Purified Components. In: Straube, A. (eds) Microtubule Dynamics. Methods in Molecular Biology, vol 777. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-252-6_14
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DOI: https://doi.org/10.1007/978-1-61779-252-6_14
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61779-252-6
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