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ABSTRACT: Cryo-electron microscopy (cryo-EM) directly images the distribution of electrostatic potential (ESP) within macromolecules, and thus can provide much more information about atomic charge than X-ray crystallography. The electron-scattering length of an isolated ion is quite different from that of the corresponding neutral atom. The difference is very large at small scattering angles where the effects of electron distributions are largest, but becomes smaller at high scattering angles where nuclear charge determines outcomes. For this reason, in cryo-EM maps that have been solved at resolutions lower than ∼2.5 Å, peaks corresponding to anions will always be less prominent than those of cations, and may even be negative. Furthermore, if a map of this kind is smeared computationally after the fact, which reduces its effective resolution, anion peaks will diminish in size, cation peaks will grow and peaks that represent uncharged atoms will remain about the same. These effects can be used to determine the sign of the charges carried by the ions associated with a macromolecule and even estimate their magnitudes. The ESP value for a cation in a cation–anion pair is smaller than the value of the cation in isolation, but the ESP value for the anion in the ionic pair is greater than the value of the anion in isolation. The experimental range of ESP values for Mg²⁺ relative to that of the closest C1′ atom is found to be between 0.57 and 1.27.