Aims
Microcrystal electron diffraction (MicroED) is becoming very appealing to solve high-resolution molecular structures for small pharmaceutical molecules and proteins without the need to grow large crystals. The novel technique of Microcrystal electron diffraction (MicroED), or 3D ED, has been developed quickly in the recent years. We started our research on MicroED at CryoEM facility of University of Wollongong from early 2021 with the aims to set up MicroED workflow and support the research society and pharmaceutical industries.
Methods
We successfully developed the MicroED workflows for both small molecules and macromolecules. Our workflow includes sample preparation, continuous-rotation data collection with Delphi and a ceta camera on Talos Arctica TEM, and data processing through DIALS. Small molecular structure solving and refinement are carried out with SHELX and Olex2. For macromolecular structure solutions, phasing, refinement and model building were performed in Phenix. The 3D model was examined and modified in COOT.
Results
We have solved acetaminophen structure to 0.77 Å, carbamazepine structure to 0.9 Å, and determined a novel organic molecule structure which couldn’t be determined by traditional methods at 0.70 Å.
Special needle-shaped lysozyme crystals were obtained for the purpose to grow nano-sized crystals. These needle-shaped crystals showed as band or ribbon shape under electron microscope about 1-1.5 µm in width and very long and thin. The lysozyme structure was solved with the resolution of 2.75 Å by merging 27 datasets. A rare space group was determined with the unit cell parameters of 31.79, 54.42, 72.72, 90.0, 99.1, 90.0, which is amazingly nearly the same as the novel polymorph of lysozyme report by Gemmi’s team in 2018.
Our MicroED workflow was fully set up in 2023 and trainings are available to academic researches and industrial specialists.