Identification of Mg²⁺ ions next to nucleotides in cryo-EM maps using electrostatic potential maps

Cryo electron microscopy (cryo-EM) can produce maps of macromolecules that have resolutions that are sufficiently high that structural details such as chemical modifications, water molecules and bound metal ions can be discerned. However, those accustomed to interpreting the electron-density maps of macromolecules produced by X-ray crystallography need to be careful when assigning features such as these in cryo-EM maps because cations, for example, interact far more strongly with electrons than they do with X-rays. Using simulated electrostatic potential (ESP) maps as a tool led us to re-examine a recent cryo-EM map of the human ribosome, and we realized that some of the ESP peaks originally identified as novel groups covalently bonded to the N7, O6 or O4 atoms of several guanines, adenines or uridines, respectively, in this structure are likely to instead represent Mg²⁺ ions coordinated to these atoms, which provide only partial charge compensation compared with Mg²⁺ ions located next to phosphate groups. In addition, direct evidence is provided for a variation in the level of 2'-O ribose methylation of nucleotides in the human ribosome. ESP maps can thus help in identifying ions next to nucleotide bases, i.e. at positions that can be difficult to address in cryo-EM maps due to charge effects, which are specifically encountered in cryo-EM. This work is particularly relevant to nucleoprotein complexes and shows that it is important to consider charge effects when interpreting cryo-EM maps, thus opening possibilities for localizing charges in structures that may be relevant for enzymatic mechanisms and drug interactions.