Methodological developments for time-resolved single particle cryo-EM (FWOAL824)

Protein synthesis and degradation, energy metabolism, signalling, processing of information-encoding polymers are facilitated and regulated by molecular machines, protein complexes that undergo significant conformational changes in time as they facilitate their function. A powerful way to gain detailed insight onto function of these proteins is to see these molecules at atomic resolution while they function. The high-resolution structures of biological macromolecules obtained by X-ray crystallography, NMR and cryo-EM have provided insights onto the way many molecular machines are constructed. Visualizing the conformational changes along the reaction trajectory at high-resolution often remains an elusive objective. The aim of my proposal is to develop methods for visualizing transient conformations of protein complexes by time-resolved single particle cryo-EM. Time-resolved cryo-EM studies structural kinetics by freeze-trapping kinetic intermediates in a biological reaction and has potential to provide atomic-resolution ‘movies’ of functioning biological complexes. Technical limitations however so far restricted widespread use and utilization of the complete potential of this technique. If successful, our approaches will allow trapping kinetic intermediates of molecular machines with millisecond time-resolution using nanoliter amounts of protein sample. The method will be applied to resolve key functional conformations of respiratory complex I and ligand-gated ion channels.

Keywords:biotechnology, kinetics

Disciplines:Other biotechnology, bio-engineering and biosystem engineering not elsewhere classified