Atomic Resolution Cryo-EM Structure of B-galactosidase
We report methods to account for radiation damage and local changes in defocus and image drift, enabling visualization of atomic resolution features in a cryo-EM density map of inhibitor-bound -galactosidase, and derivation of atom-specific measures of local flexibility of the bound inhibitor using constrained molecular dynamics simulations.
– Improved methods for tracking particle movement during the electron exposure.
– Atomic resolution cryo-EM map of inhibitor bound -galactosidase enzyme.
– Delineation of spherical contours of non-hydrogen atoms and visualization of active site.
– Quantitative evaluation of inhibitor flexibility using constrained molecular dynamics simulations.
The advent of direct electron detectors has enabled the routine use of single particle cryo-EM approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for inhibitor-bound -galactosidase where the ordered regions are resolved at a level of detail seen in X-ray maps at ~ 1.5 Å resolution. We show that this map can be used to compute atom-specific measures of local flexibility of the bound inhibitor PETG, providing a general approach for structure-guided improvement of inhibitor design with atomic resolution cryo-EM.