Structure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron Microscopy to 1.42Å Resolution

Single particle Cryo-Electron microscopy (Cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution at which individual atoms, including hydrogen atoms, can be resolved. Thus Cryo-EM allows not only unprecedented detail regarding the structural architecture of complexes but also a better understanding surrounding their chemical states. In this study we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5Å or better which so far has only been achieved for the iron carrier ferritin. Using state of the art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48-hour microscope session. In addition to water molecules and ligands involved in AaLS function, we can observe positive density for ∼50% of hydrogen atoms. A small improvement in resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.