Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome

Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC beta II binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC beta II interacts with RACK1, a seven-bladed beta-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC beta II/RACK1 interaction and its role in translation. (C) 2012 Elsevier Inc. All rights reserved.