Hydrogens and hydrogen-bond networks in macromolecular MicroED data

Abstract

Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that scattered electrons carry informing about the charged state of atoms and can provide strong contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for drug discovery and electron microscopy can enable such visualization. Using subatomic resolution MicroED data obtained from triclinic hen egg-white lysozyme, we identified hundreds of individual hydrogen atom positions and directly visualize hydrogen bonding interactions and the charged states of residues. Over a third of all hydrogen atoms are identified from strong difference peaks, the most complete view of a macromolecular hydrogen network visualized by electron diffraction to date. These results show that MicroED can provide accurate structural information on hydrogen atoms and non-covalent hydrogen bonding interactions in macromolecules. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering and understanding the hydrogen bonding interactions underlying protein structure and function.

Electron-counting MicroED data with the K2 and K3 direct electron detectors

“…For these reasons, previous MicroED studies using direct electron detectors such as the Falcon 3 (Thermo Fisher) and the DE64 (Direct Electron) for protein structure determination relied on integrating/linear mode rather than counting (Hattne et al ., 2019; Takaba et al ., 2021). It is, however, possible to collect electron-counted MicroED data by significantly lowering the exposure, and this was recently shown to provide data of sufficiently high quality for ab initio macromolecular structure determination using the Falcon 4 (Thermo Fisher) direct electron detector (Martynowycz et al ., 2022; Clabbers et al ., 2022). These results are encouraging, however MicroED data collection using direct electron counting is not yet mainstream despite the availability of these cameras in cryo-EM laboratories.…”