AbstractElectron microscopy can provide accurate, high‐resolution images of the distribution of electrostatic potential (ESP) in biological macromolecules. Careful examination of ESP maps that have been published for peptides and proteins at resolution ranging from 1.0 Å to 2.9 Å reveals that the negative charges of carboxylate groups have a profound effect on their appearance. It is clear that investigators must take the negative features in their experimental ESP maps into account when modeling the conformations of Asp and Glu side chains and those of the residues that surround them.