Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117-125

Howard Hughes Medical Institute, Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454, USA.
Journal of Structural Biology (Impact Factor: 3.37). 02/2007; 157(1):117-25. DOI: 10.1016/j.jsb.2006.05.004
Source: PubMed

ABSTRACT The refinement of three-dimensional reconstructions and correction for the contrast transfer function of the microscope are important steps in the determination of macromolecular structures by single particle electron microscopy. The algorithms implemented in the computer program FREALIGN are optimized to perform these tasks efficiently. A general overview and details on how to use FREALIGN are provided. The program is free and available for download on the author's web page.

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Available from: Nikolaus Grigorieff, Jul 29, 2015
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    • "In single particle EM, two dimensional (2D) projection images of biological specimens are recorded in an electron microscope, their relative orientations are determined using one of a number of alignment algorithms, and finally one or more 3D reconstructions are calculated (Frank et al., 1996; Van Heel et al., 1996; Marabini et al., 1996; Grigorieff, 2007; Tang et al., 2007; Scheres, 2012). With favourable datasets (high signal-to-noise, even particle distributions , homogeneous conformation, etc.), iterative refinement of the orientations assigned to each particle image will converge to the true 3D density map. "
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    ABSTRACT: Determining the structure of a protein complex using electron microscopy requires the calculation of a 3D density map from 2D images of single particles. Since the individual images are taken at low electron dose to avoid radiation damage, they are noisy and difficult to align with each other. This can result in incorrect maps, making validation essential. Pairs of electron micrographs taken at known angles to each other (tilt-pairs) can be used to measure the accuracy of assigned projection orientations and verify the soundness of calculated maps. Here we establish a statistical framework for evaluating images and density maps using tilt-pairs. The directional distribution of such angular data is modelled using a Fisher distribution on the unit sphere. This provides a simple, quantitative and easily comparable metric, the concentration parameter κ , for evaluating the quality of datasets and density maps that is independent of the data collection and analysis methods. A large κ is indicative of good agreement between the particle images and the 3D density map. For structure validation, we recommend κ>10κ>10 and a p-value<0.01p-value<0.01. The statistical framework herein allows one to objectively answer the question: Is a reconstructed density map correct within a particular confidence interval?
    Journal of Structural Biology 08/2014; 187(2). DOI:10.1016/j.jsb.2014.06.006 · 3.37 Impact Factor
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    • "A total of 2,965 particles were picked by hand using Ximdisp (Smith, 1999). CTFs were estimated using CTFFIND3 (Mindell and Grigorieff, 2003), boxed particle densities were floated and normalized using BOXIMAGE (Crowther et al., 1996), and 3D maps were calculated using Frealign (Grigorieff, 2007). Part of a typical micrograph is shown in Figure 1A. "
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    ABSTRACT: Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated enzyme formation mutants. To understand the nature of this activation, we have determined by electron cryo-microscopy the structure of the complex between β-gal and the antibody scFv13R4. Our structure localizes the scFv13R4 binding site to the crevice between domains 1 and 3 in each β-gal subunit. The mutations that scFv13R4 counteracts are located between the antibody binding site and the active site of β-gal, at one end of the TIM-barrel that forms domain 3 where the substrate lactose is hydrolyzed. The mode of binding suggests how scFv stabilizes both the active site of β-gal and the tetrameric state.
    Structure 03/2014; 22(4). DOI:10.1016/j.str.2014.01.011 · 6.79 Impact Factor
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    • "Images presented into unresolved class averages were excluded for further analysis. The three-dimensional reconstruction and structure refinement was iteratively done using FREALIGN program (Grigorieff, 2007) and applying D4 symmetry. For IMPDHpa, initial models were derived from our resolved atomic structures, which were converted to electron density map and lowpass filtered at 50 A ˚ . "
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