3D electron diffraction investigation on Hen Egg-White Lysozyme crystals

New perspectives have been opened in recent years on the crystallographic analysis of submicrometric protein crystals by electron diffraction (ED), commonly referred as MicroED or 3D ED. This technique exploits the strong interaction of a parallel beam of high energy electrons with matter which is able to produce accurate diffraction patterns from crystals of a few hundred of nanometers. Despite stimulating results obtained on different protein crystals, many efforts are still needed in order to improve technical aspects of the ED analysis, as the preparation of good working samples and the spatial resolution for diffraction collections. The analysis of nanocrystals by using a precession­assisted electron nanobeam together with STEM imaging has demonstrated to provide accurate diffraction data in this regard, especially for the investigation of beam sensitive samples because of the small illuminated area in comparison to the generally performed continuous rotation data collections. To evaluate such ED methodology on macromolecular specimens, we used protein crystals of the Hen Egg White Lysozyme as first case study. Crystals were deposited on carbon­coated grids, followed by blotting and vitrification by plunging the grids into liquid ethane. Diffraction data collections on a new monoclinic polymorph and on the known tetragonal form of Lysozyme were obtained using precession of the electron nanobeam and have led to the solution via molecular replacement and the refinement of both structures. Notably, findings on the novel monoclinic polymorph suggest a new path to characterize the molecular dynamics associated to protein crystal growth. The promising results obtained will be presented together to an informative picture of ED and its future perspectives.