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 speci fi cally designed for the image processing of biological structures imaged with electron or X-ray microscopy. We illustrate the image processing work fl ow 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
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