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.
Key wordsSingle particle analysis Electron microscopy Image processing 3D reconstruction Workflows
C.O.S. Sorzano is a recipient of a Ramón y Cajal fellowship awarded by the Spanish Ministry of Science and innovation (Micinn). J. Vargas and J. Otón are Juan de la Cierva fellows (Micinn). This work has been partly funded by grants CSD2006-0002 (Spanish Ministry of Education), ACI2009-10220 (Micinn), BIO2010-16566 (Micinn), and PIF08-020-2 (CSIC).
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