Semiautomatic, high-throughput, high-resolution protocol for three-dimensional reconstruction of single particles in electron microscopy.

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.