High-throughput automated molecular replacement for small-molecule MicroED data

Interest in Microcrystal Electron Diffraction (MicroED) for structural characterization of both proteins and small molecules has grown significantly since the method’s inception 12 years ago. While ab initio phasing methods remain the gold standard for small molecule samples MicroED data, radiation beam damage during data collection and poor crystallinity of the nanocrystalline sample can make this method unfeasible—particularly for challenging molecules that exhibit inherent flexibility or contain large substituents. Molecular replacement (MR) is the most commonly used phasing method for protein MicroED data and can circumnavigate issues related to diminished data quality. However, its application to small molecules has been limited due to the lack of diverse methods for efficiently sampling the conformational landscape of small molecules. Herein, a high-throughput, automated molecular replacement method has been developed to solve a novel MicroED structure of corilagin, a macrocyclic gallotannin with pharmaceutical relevance, which was unable to be solved with ab initio phasing. The method was validated against three other similar macrocycles (paritaprevir-α, paritaprevir-β, and grazoprevir) at varying data resolution limits (1.0, 1.2, 1.4, 1.5, 1.6, 1.8, and 2.0Å). At all these resolutions for all three structures, the developed method was successful and produced solutions with R-factors and RMSD values within acceptable limits of the ab initio solved structure. The automated method presented here is anticipated to find uses for larger and more complex molecules — including large peptides, toxins and natural products — which often yield diffraction data with resolutions poorer than 1.0 Å.