If Gel and Mass Spec Don't Help, Use cryo-EM to Characterise your Specimen

Abstract

Cryo-EM has proven to be efficient in obtaining high-res maps for purified and well-characterized specimen. But how about heterogenous complexes with unknown associated moieties?We applied cryo-EM to the pool of free ribosomal subunits in human mitochondria. Intensive classification generated 3.5 A resolution map from 60% of the total particles. Comparison of the map with that of the intact human mitoribosome revealed disordered rRNA at the interface and presence of a silencing factor. This suggested that the subset of particles isolated directly from the cellular compartment represents mitoribosome assembly intermediate.Further inspection revealed extra protein density of an unknown origin bound to the silencing factor, namely ‘silencing factor binding protein’. The model clearly suggests a function for this newly discovered protein in mitoribosome biogenesis. Therefore we present first high-res model of mitoribosome assembly intermediate and show that cryo-EM can be used to identify new proteins, propose their function and provide a basis for designing new biochemical experiments to further investigate the discovered mechanisms.