Stabilization of Ribosomal RNA of the Small Subunit by Spermidine in Staphylococcus aureus

Cryo-electron microscopy is now used as a method of choice in structural biology for studying protein synthesis, a process mediated by the ribosome machinery. In order to achieve high-resolution structures using this approach, one needs to obtain homogeneous and stable samples, which requires optimization of ribosome purification in a species-dependent manner. This is especially critical for the bacterial small ribosomal subunit that tends to be unstable in the absence of ligands. Here, we report a protocol for purification of stable 30 S from the Gram-positive bacterium Staphylococcus aureus and its cryo-EM structures: in presence of spermidine at a resolution ranging between 3.4 and 3.6 angstrom and in its absence at 5.3 angstrom. Using biochemical characterization and cryo-EM, we demonstrate the importance of spermidine for stabilization of the 30 S via preserving favorable conformation of the helix 44.

Automated summary

Cryo-electron microscopy is now a preferred method for studying protein synthesis through ribosome machinery. To achieve high-resolution structures, optimization of ribosome purification in a species-dependent manner is necessary. This study demonstrates the importance of spermidine in stabilizing the 30S ribosomal subunit by preserving favorable conformation of helix 44.