Atomic Resolution Cryo-EM Structure of beta-Galactosidase

The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (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 beaminduced drift, and the implementation of a datadriven 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 beta-galactosidase bound to the inhibitor phenylethyl beta-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at similar to 1.5 angstrom resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the noncovalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.