Cryo-electron tomography of microtubules assembled in vitro from purified components.
Methods Mol Biol
; 777: 193-208, 2011.
Article
en En
| MEDLINE
| ID: mdl-21773930
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Microscopía por Crioelectrón
/
Tomografía con Microscopio Electrónico
/
Microtúbulos
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2011
Tipo del documento:
Article
País de afiliación:
Francia
Pais de publicación:
Estados Unidos