title: Semiautomatic, high-throughput, high-resolution protocol for three-dimensional reconstruction of single particles in electron microscopy.
abstract: In this chapter we describe the steps needed for reconstructing the three-dimensional structure of a macromolecular complex starting from its projections collected in electron micrographs. The concepts are shown through the use of Xmipp 3.0, a software suite specifically designed for the image processing of biological structures imaged with electron or X-ray microscopy. We illustrate the image processing workflow by applying it to the images of Bovine Papilloma virus published in Wolf et al. (Proc Natl Acad Sci USA 107:6298-6303, 2010). We show that in the case of high-quality, homogeneous datasets with a priori knowledge about the initial volume, we can have a high-resolution 3D reconstruction in less than 1 day using a computer cluster with only 32 processors.
authors.affiliation: Biocomputing Unit, National Center of Biotechnology (CSIC), Universidad Autónoma de Madrid, Madrid, Spain. coss@cnb.csic.es
authors.forename: Carlos Oscar
authors.initials: CO
authors.lastname: Sorzano
authors.forename: J M
authors.initials: JM
authors.lastname: de la Rosa Trevín
authors.forename: J
authors.initials: J
authors.lastname: Otón
authors.forename: J J
authors.initials: JJ
authors.lastname: Vega
authors.forename: J
authors.initials: J
authors.lastname: Cuenca
authors.forename: A
authors.initials: A
authors.lastname: Zaldívar-Peraza
authors.forename: J
authors.initials: J
authors.lastname: Gómez-Blanco
authors.forename: J
authors.initials: J
authors.lastname: Vargas
authors.forename: A
authors.initials: A
authors.lastname: Quintana
authors.forename: Roberto
authors.initials: R
authors.lastname: Marabini
authors.forename: José María
authors.initials: JM
authors.lastname: Carazo
mesh_terms: D000818:Animals; D001331:Automation; D002213:Capsid / Q000648:ultrastructure; D002417:Cattle; D052687:Deltapapillomavirus / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D013997:Time Factors