Due to its charge, electron interacts strongly with matter. Electron crystallography can thus be carried out on nanometer-sized crystals that contain fewer unit cells than crystals typically required for X-ray or neutron crystallography. Moreover, the resulting Coulomb potential map can provide additional information not available in the electron density map, such as the valence of the ions [1]. Although electron crystallography has successfully resolved three-dimensional structures of proteins from vitrified crystals, its widespread use as a structural biology tool has been limited. One of the reasons is the fragility of protein crystals, which can be easily damaged by mechanical stress and temperature changes, etc. Liquid phase transmission electron microscopy may present an alternative to vitrification and cryo-electron microscopy for electron crystallography [2].
In this work, nanocrystals of lysozyme with a size range suitable for electron diffraction were grown using the batch technique. These nanocrystals were then encapsulated by graphene membranes in their mother liquor on the electron microscopy grids. Graphene is a two-dimensional material with exceptional mechanical strength and excellent thermal and electrical conductivities. These properties make them very suitable as a substrate for samples in electron microscopy studies. Besides the ability to seal the sample solution hermetically, the graphene layers further protect the sample against radiation damage from the imaging electrons in the microscopy.
Electron diffraction experiments on the encapsulated crystals were carried out on the F20 microscope of the IBS/ISBG electron microscopy platform at room temperature with the standard room temperature sample holder. Using the low-dose technique routine for imaging biological samples and the hybrid pixel detector installed on the F20 microscope, diffraction spots of up to 3 Å resolution were obtained. Indexing of the diffraction patterns is also possible thanks to a pattern matching algorithm.
Nanosized hydrates are important in many fields of science and technology, including energy conversion and storage, and biomedicine [3]. The technique employed here for room temperature electron crystallography can potentially be applied to many other organic or inorganic hydrates in different research areas.
M. Spano, D. Housset, W.L. Ling (IBS)
[1] T.B. Blum, D. Housset, M.T.B. Clabbers, E. van Genderen et al. (2021). Acta Cryst. D Struct. Biol., 77, 75-85..
[2] L. Kong, J. Liu, M. Zhang, Z. Lu et al. (2023). Nat Commun., 14, 5641.
[3] S. Plana-Ruiz, A. Gómez-Pérez,M. Budayova-Spano, D.L. Foley et al. (2023). ACS Nano, 17, 24, 24802-24813
[4] C. Labbez, L. Bouzouaid, A.E.S. van Driessche, W.L. Ling et al. (2023). Cem. Concr. Res., 173, 107299.
Figure: Lysozyme nanocrystal in crystallization solution is encapsulated in graphene layers for electron diffraction at room temperature. Reflections up to 3 angstroms are obtained.
