By contrast, the cryo-EM experiment yields both amplitudes and phases directly from images and experimentally determined EM density can therefore be used as a constant minimization target ( Grigorieff et al., 1996 Unwin, 2005 Yonekura et al., 2003). As a result, crystallographic refinement successively improves map interpretability throughout the refinement procedure. Coordinate refinement in X-ray crystallography typically depends on initial phase estimates that are iteratively improved ( Agarwal and Isaacs, 1977 Grosse-Kunstleve et al., 2002 Lunin et al., 2002 Read, 1986 Wilson, 1942). Sophisticated protocols for map interpretation as well as parameterization and validation of coordinate refinement in X-ray crystallography exist and these procedures have been adapted to work with cryo-EM maps ( Amunts et al., 2014 Brown et al., 2015 Fromm et al., 2015 Hoffmann et al., 2015 Wang et al., 2014). The building and refinement of atomic models thus present a critical step in the structure determination process and their accuracy depends on the quality of the EM density. Atomic models are frequently used by biologists without expert knowledge to interrogate function based on mechanistic hypotheses inferred from the structure. At this resolution, the reconstructed EM density maps contain sufficient detail to interpret the structure using atomic models. Major improvements in detector technology ( Faruqi and Henderson, 2007 McMullan et al., 2016) and associated computational procedures recently transformed single-particle cryo-EM that since has been producing a plenitude of near-atomic resolution structures from specimens of lower symmetry ( Allegretti et al., 2014 Amunts et al., 2014 Bai et al., 2013) and lower molecular weight than previously deemed possible ( Bai et al., 2015 Liao et al., 2013 Merk et al., 2016). Electron cryo-microscopy (cryo-EM) has been used as a method to visualize biological macromolecules in their native-hydrated state for more than three decades ( Adrian et al., 1984).
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