Puzzling structures enlightened. A structural survey of large protein complexes

PhD ceremony: Mr. J.B. Bultema, 13.15 uur, Academiegebouw, Broerstraat 5, Groningen

Thesis: Puzzling structures enlightened. A structural survey of large protein complexes

Promotor(s): prof.dr. E.J. Boekema

Faculty: Mathematics and Natural Sciences

This thesis describes the results of structural characterization of large proteins complexes by single particle electron microscopy. The main aim of this project was to determine the structures of multi-protein complexes and to localize subunit positions within these multi-protein complexes. This information provides insights to better understand protein-protein interaction within complexes. One chapter describes the structural characterization of isolated mitochondrial supercomplexes. Projection maps of such supercomplexes were analysed. Based on our assignment of individual complexes in the supercomplexes we propose a structural model for the largest supercomplex. Two chapters describe several aspects of Vipp1 from Synechocystis PCC 6803. We showed that Vipp1 forms stable dimers and higher-ordered oligomers. Vipp1 is localized in the cytoplasm as well as at the cytoplasmic and thylakoid membrane. The simultaneous existence of multiple types of rings is extraordinary and suggests a special function of Vipp1. The second chapter describes a comparison with Vipp1’s bacterial homologue Phage Shock Protein A (PspA). Both proteins have similar secondary structures and form large homooligomeric rings but their ring dimensions differ significantly. Furthermore, we suggest a spatial structural model of the observed Vipp1 rings.The last chapter describes the biochemical and biophysical characterization of a ribonucleoprotein complex called Cascade. This multi-protein complex is part of the CRISPR immune system in prokaryotes which utilizes small guide RNAs to neutralize invading viruses and plasmids. We present the composition, protein stoichiometry and low-resolution structure of Cascade and show how it recognizes double-stranded DNA targets.