We study the allosteric regulation of the essential enzyme ribonucleotide reductase (RNR) in a wide variety of organisms using a range of structural biology techniques, including X-ray crystallography, small-angle X-ray scattering and cryo-electron microscopy. RNR is a central enzyme for all but a few living organisms, as it catalyses an essential step in the synthesis of the deoxyribonucleotide building blocks necessary for DNA synthesis and repair. At present our main focus is on the role of protein oligomerisation in regulation of the overall activity of the enzyme, which most frequently consists of two subunits, a catalytic subunit and a radical-generating subunit. The binding of dATP to a small, 100-residue domain in the large subunit, called the ATP-cone, causes it to build various forms of oligomer (tetramers, hexamers etc.) that have in common that they prevent the radical-generating subunit from binding in a productive manner. However the manner in which this steric inhibition is achieved vary dramatically from organism to organism, and the forms of oligomerisation are several and diverse. We are interested in the evolutionary development of the ATP-cone domain. Our studies have suggested that such an understanding of the differences in activity regulation between organisms could lead to the development of specific inhibitors that could switch off pathogenic RNRs while not interfering with the essential human enzyme. Thus we plan to collect cryoEM data from RNRs from several organisms and compare them to crystal structures and SAXS data from RNRs of other organisms that we and others have already collected. The complexes formed are usually in the range 200-500 kDa, which makes them ideal for cryoEM.
The first sample that we have focused on is the tetrameric, dATP-inhibited ribonucleotide reductase large subunit from the human pathogen Chlamydia trachomatis. Several hundred micrographs have been collected on the Talos Arctica microscope at SciLifeLab in Stockholm, and processing is underway (see "Resource Usage").