Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial (Escherichia coli) and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick base pairs and characterized by a broad minimum of the electrostatic potential in the deep (major) groove. The bacterial loop E initial geometry was taken from the crystal structure (URL064). The starting structure of spinach chloroplast Loop E was modeled using isostericity principles and the simulations refined the geometries of the three non-Watson-Crick base pairs that differ from the consensus bacterial sequence. The deep groove of loop E motifs provides unique sites for cation binding. Binding of Mg²⁺ rigidifies Loop E and stabilizes its major groove at an intermediate width. Mg²⁺ cations observed by X-ray diffraction in the bacterial Loop E are stable in the simulations. They do not bind directly at the most negative sites, as these are buried too deeply inside the groove. The spinach chloroplast Loop E sequence tends to relocate the Mg²⁺ cations compared to the bacterial Loop E cation distribution. In the absence of Mg²⁺, the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations and a wide range of deep groove widths, depending on the base sequence and the counterion distribution. The Na⁺ cations penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with that of Escherichia coli (which is almost identical to the bacterial consensus). Structures with very narrow deep groove essentially collapse around a string of Na⁺ cations with very long coordination times. The Loop E non-Watson-Crick base pairing is complemented by a number of highly specific hydration sites ranging from simple water bridges to complex hydration pockets involving up to five hydration centers hosting 2 to 3 long-residing water molecules. The ordered hydration is intimately connected with local conformational variations of the RNA molecule.