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Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If, as is typically the case, the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the grid cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that this method could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop crystallization approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the COVID-19 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting preferred orientations, unlocking new possibilities for structure determination by MicroED.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus infection. Its structures had been elusive to the public until recently when one of the crystal forms was solved by MicroED. In this work, we report the MicroED structures of two distinct polymorphic crystal forms of paritaprevir from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropylsulfonamide and methylpyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the NS3/4A serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acylsulfonamide inhibitors to the NS3/4A serine protease. In addition, this also demonstrate the opportunity of deriving different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles which have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material, and that different conformations of flexible compounds can be derived from the same experiment. These results will have impact on contemporary drug discovery as well as natural product exploration.