Proper assembly and maturation of ribosomal subunits are critical processes that ensure functional activity, translation efficiency, and fidelity of the ribosome. The GTP-binding protein YsxC is found in many bacteria and is one of the protein factors involved in maturation of the large ribosomal subunit. Immature ribosomal intermediates, designated 45S subunits, are known to accumulate within the cell in the absence of YsxC. The 45S subunits cannot associate with the small ribosomal subunit and thus fail to form ribosomes capable of their necessary functions. A deletion of the ysxC gene is lethal in Staphylococcus aureus. The mechanism of YsxC interactions with the S. aureus ribosome remains to be elucidated. A protocol was devised to isolate, purify, and assembly the YsxC protein complex with the 50S subunit of the S. aureus ribosome. A sample obtained according to the protocol proved suitable for data collection by transmission cryo-electron microscopy.