System Preparation. The simulated complex was prepared with Pymol 21 by superimposing the cryo-EM structure of the complex formed between B. Subtilis 50S (pre-5S) ribosomal RNA subunit and the M5 enzyme 9 (PDB: 6TPQ), with the X-ray structure 10 (code PDB: 6Z2B) of uncomplexed M5 Toprim domain (chain B)-which contains two Mg 2+ ions in the active site. All water molecules resolved in the X-ray structure (in particular in the Mg 2+ hydration shell) were retained, and the mutation of Asp58 to alanine was reversed.