Stefan Pfeffer Welcome to the Pfeffer Lab!

Co-translational folding and maturation of proteins require an intricate network of folding chaperones and processing enzymes that act on the growing nascent protein in a co-translational manner and co-purify with polysomal complexes. Structural information on ribosome-nascent chain-chaperone complexes is sparse, because the involved interactions are mostly transient, labile and possibly highly flexible for chaperones binding exclusively to the growing nascent protein. This renders the involved assemblies inaccessible to classical reductionist structural biology approaches that rely on extensive biochemical purification and require conformationally homogenous particle populations for averaging.

We consequently pursue a different approach and image these processes using cryo electron tomography (cryo-ET)-based strategies, which can reveal the three-dimensional arrangement of individual macromolecules even in crowded native microenvironments at molecular resolution and therefore render extensive biochemical purification unnecessary. This approach allows us to analyze the three-dimensional spatial distribution of ribosomes, chaperones and processing enzymes for individual native polysomal assemblies under conditions that preserve the labile and transient interactions governing co-translational protein folding and maturation. We study defined polysomal assemblies engaged in the synthesis of model substrates in various organisms, visualizing these assemblies in both a non-cellular context and in sections of vitrified unperturbed cells obtained using focused ion beam (FIB) milling.

In order to gain the most detailed structural insights possible into co-translational protein folding and maturation, we also innovate cryo-ET based data acquisition strategies and image processing schemes, optimizing them for high resolution. In particular, we integrate complementary information from different cryo-EM approaches to gain information on the three-dimensional architecture of the specimen while retaining high-resolution signal for structure determination.