Sub-Angstrom Structure Determination of Organic Molecules at Room Temperature Using 100 KeV Serial Electron Diffraction

Finding the atomic structure of materials and molecules is crucial for understanding their properties and functions in material and life sciences. Crystallography is commonly used to determine the structure of proteins, organic and inorganic molecules, and other materials by analyzing the diffraction patterns formed when X-rays or electron beams pass through them. While X-ray crystallography has been the go-to method for many years, it requires large, well-ordered crystals, which can be challenging to obtain for certain materials like membrane proteins [1]. Single-particle cryo-electron microscopy (Cryo-EM) has also been successful in determining the high-resolution structure of many proteins. However, this method is typically unable to solve the structures of small proteins [2]. On the other hand, serial femtosecond crystallography using x-ray free electron laser (XFEL) sources has succeeded in determining the structures of submicron-sized crystals [3]. However, XFEL sources are billion-dollar machines with high operation costs, which limit their applicability. Microcrystal electron diffraction (MicroED) has proven effective in determining the structures of biomaterials and small molecules over the past decade [4]. However, dose accumulation from high energy electrons during the rotation of the crystal limits the application of the MicroED for beam sensitive materials [5]. Therefore, a Cryo-EM method is typically employed to mitigate beam damage. While Cryo-EM instruments are much cheaper than XFEL sources, they still entail multimillion-dollar investments with the added cost of special room preparation and expensive detectors. Thus, the structure determination of sub-micron-sized crystals remains relatively costly. Recently, serial electron diffraction (SerialED) crystallography has been developed and applied successfully for structure determination of small molecules [6, 7] and proteins [8]. In this approach, diffraction patterns are sequentially collected from thousands of nanocrystals, minimizing beam damage by spreading the electron dose over many nanocrystals. In our study, we demonstrated the feasibility of determining the structure of a beam-sensitive organic molecule with sub-angstrom resolution on a 100 kV electron microscope at room temperature. The low-kV electron microscopes without Cryo typically cost less than a million dollars, significantly reducing the expense of structure determination. Moreover, the beam damage on organic samples is generally lower at lower electron energies. We utilized a Hitachi HT7700 transmission and scanning transmission electron microscope (TEM/STEM) equipped with an X-Spectrum Amber 750K detector to solve the structure of carbamazepine, a beam-sensitive organic small molecule [9]. The diffraction data is collected sequentially in the STEM mode using the Azorus package (Hitachi High-Tech Canada Inc.) by defining a custom scan pattern to map the crystal coordinates. Fig. 1-A illustrates the carbamazepine crystals, with approximately 2000 successfully indexed diffraction patterns collected from selected points of different orientations of carbamazepine crystals (Fig. 1-B). Room temperature MicroED yielded a 1.0 Å resolution structure [10] while Cryo-EM MicroED resulted in a 0.85 Å structure for carbamazepine [11]. In our work, we achieved a 0.7 Å resolution structure of carbamazepine at room temperature using SerialED with 100 keV electrons. Fig. 2-A displays the solved structure of carbamazepine with the electron density map. We determined the resolution from the signal-to-noise ratio (I/σ(I)) of the diffraction data, where it falls below 2, as shown in Fig. 2-B. In conclusion, SerialED offers a cost-effective means of attaining sub-angstrom resolution structures of beam-sensitive materials using a readily available electron microscope. With advancements in nanofluidic chips and automation in data collection and processing, SerialED can become a high-throughput method for in situ structure determination of various materials, including organic molecules, proteins, etc. [12]. (A) Annular darkfield scanning TEM image of carbamazepine crystals. The diffraction patterns are collected sequentially from the selected points. (B) The series of diffraction patterns collected from the sample. (A) The structure of carbamazepine with the electron density map overlay. (B) The signal-to-noise ratio plot over the inverse of d-spacing that shows the resolution cut off at I /σ(I) = 2.