In vitro Reconstitution of the S. aureus 30S Ribosomal Subunit and RbfA Factor Complex for Structural Studies

Ribosome-binding factor A (RbfA) from Staphylococcus aureus is a cold adaptation protein that is required for the growth of pathogenic cells at low temperatures (10-15°C). RbfA is involved in the processing of 16S rRNA, as well as in the assembly and stabilization of the small 30S ribosomal subunit. Structural studies of the 30S–RbfA complex will help to better understand their interaction, the mechanism of such complexes, and the fundamental process such as 30S subunit assembly that determines and controls the overall level of protein biosynthesis. This article describes protocols for preparation of RbfA and the small 30S ribosomal subunits and reconstitution and optimization of the 30S–RbfA complex to obtain samples suitable for cryo-electron microscopy studies.