The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination

Abstract

In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10^-15 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.

Figure 1

Process of applying MicroED to small molecule structural analysis. Here commercial progesterone (1) was analyzed, and an atomic resolution structure was determined at 1 Å resolution. Grid holes are 1 μm in diameter.

Figure 2

Different types of small molecules solved by MicroED. (A) Several pharmaceuticals, vitamins, commercial natural products, and synthetic samples resolved through MicroED. (B) Example of an amorphous film utilized in this study leading to 1 Å resolution data. (C) Protons could be observed for several compounds through MicroED. Green density are F_o – F_c maps showing positive density belonging to hydrogen atoms of the molecule.

Figure 3

Identification of compounds from heterogeneous mixtures. An EM grid was prepared as above with biotin, brucine, carbamazepine, and cinchonine powders mixed together. All four compounds identified by unit cell parameters using MicroED data from within the same grid square. All structures were solved to ∼1 Å resolution. Grid holes are 2 μm in diameter.