3D Electron Diffraction Methods for Crystal Structure Determination - From Materials to Proteins

Hongyi Xu, Department of Materials and Environmental Chemistry, Stockholm University, Sweden

Knowing the 3D atomic structures of materials or biomolecules is crucial for understanding their functions. X-ray diffraction is currently the most important technique for determination of 3D atomic structures, but requires large crystals which are often difficult to obtain. Electrons, similar to X-rays and neutrons, are powerful source for diffraction experiments¹. Due to the strong interactions between electrons and matter, crystals that are considered as powder in X-ray crystallography can be treated as single crystals by 3D electron diffraction methods^2,3. This enables structure determination of materials and organic molecules from micron- to nanometer-sized 3D crystals that are too small for conventional X-ray diffraction. Furthermore, by taking the advantages of the unique properties of electron scattering, it is possible to determine the charge states of atoms/ions4 and the absolute structure of chiral crystals^5,6.

Over the past decades, a number of 3D ED methods have been developed for structure determination. At the early stages of 3D ED method development, tilting of the crystal was done manually, while diffraction patterns were collected on negative film. It could take years before sufficient data were obtained and processed in order to determine the crystal structure. The computerization of TEMs and the development of CCD detectors allowed software to be developed that can semi-automatically collect 3D ED data in less than an hour (ie. rotation electron diffraction, RED² in Zou’s lab). Thanks to the recent advancement in CMOS and hybrid detector technology, it is now feasible to collect diffraction data in movie mode while continuously rotating the crystal (continuous rotation election diffraction, cRED, also known as MicroED in structural biology). Benefiting from these technological advances, structure determination can now be accomplished within a few hours. Recently, fully automated serial rotation electron diffraction data collection and processing has been realized by our group⁷.

By using 3D ED / MicroED methods, we have solved more than 150 novel crystal structures of small inorganic compounds (including zeolite8, MOF9, COF10 and minerals) and organic molecules (pharmaceuticals11, small organic molecules, peptides and proteins^12,13) in the past 7 years. Recently, we have solved two novel protein structures^13,14 by MicroED and shown that it is feasible to use the method for structure based drug discovery¹⁵. We aim to further improve these methods, develop new methods¹⁶ and more importantly spread them to labs around the world.

References

1. Zou, X., Hovmöller, S. & Oleynikov, P. Electron crystallography: electron microscopy and electron diffraction. (Oxford University Press, 2011).
2. Wan, W., Sun, J., Su, J., Hovmöller, S. & Zou, X. Three-dimensional rotation electron diffraction: software RED for automated data collection and data processing. J. Appl. Crystallogr. 46, 1863–1873 (2013).
3. Kolb, U., Gorelik, T. E., Mugnaioli, E. & Stewart, A. Structural Characterization of Organics Using Manual and Automated Electron Diffraction. Polym. Rev. 50, 385–409 (2010).
4. Yonekura, K., Kato, K., Ogasawara, M., Tomita, M. & Toyoshima, C. Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges. Proc. Natl. Acad. Sci. 112, 3368–3373 (2015).
5. Brázda, P., Palatinus, L. & Babor, M. Electron diffraction determines molecular absolute configuration in a pharmaceutical nanocrystal. Science 364, 667–669 (2019).
6. Xu, H. & Zou, X. Absolute structure, at the nanoscale. Science 364, 632–633 (2019).
7. Wang, B., Zou, X. & Smeets, S. Automated serial rotation electron diffraction combined with cluster analysis: an efficient multi-crystal workflow for structure determination. IUCrJ.
8. Wang, Y., Yang, T., Xu, H., Zou, X. & Wan, W. On the quality of the continuous rotation electron diffraction data for accurate atomic structure determination of inorganic compounds. J. Appl. Crystallogr. 51, 1094–1101 (2018).
9. Wang, B. et al. A porous cobalt tetraphosphonate metal-organic framework: accurate structure and guest molecule location determined by continuous rotation electron diffraction. Chem. - Eur. J. (2018) doi:10.1002/chem.201804133.
10. Ding, H. et al. An AIEgen-based 3D covalent organic framework for white light-emitting diodes. Nat. Commun. 9, (2018).
11. Wang, Y. et al. Elucidation of the elusive structure and formula of the active pharmaceutical ingredient bismuth subgallate by continuous rotation electron diffraction. Chem. Commun. 53, 7018–7021 (2017).
12. Xu, H. et al. A Rare Lysozyme Crystal Form Solved Using Highly Redundant Multiple Electron Diffraction Datasets from Micron-Sized Crystals. Structure 26, 667-675.e3 (2018).
13. Xu, H. et al. Solving a new R2lox protein structure by microcrystal electron diffraction. Sci. Adv. 5, eaax4621 (2019).
14. Clabbers, M. T. B. et al. MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography. Nat. Commun. 12, 2578 (2021).
15. Clabbers, M. T. B., Fisher, S. Z., Coinçon, M., Zou, X. & Xu, H. Visualizing drug binding interactions using microcrystal electron diffraction. Commun. Biol. 3, 417 (2020).
16. Zhao, J. et al. A simple pressure-assisted method for MicroED specimen preparation. Nat. Commun. 12, 5036 (2021).