The staff shadowing program awards up to 20 staff per round. It aims to foster new skills, transfer knowledge and facilitate collaborations for staff in Microscopy Australia nodes and linked labs. The successful applicants gain more research experience to provide enhanced support to Microscopy Australia users.
I was first awarded a shadowing opportunity back in November 2019 to travel internationally in May 2020. Before this could happen however, the Canberra region saw massive bushfires and a hailstorm that damaged the whole ANU campus. When Covid19 started, I was forced to re-think my options for training due to the closure of international borders.
We jump forward in time to 2023 and with all borders open again for international travel, I got another chance to learn new things. With the arrival of the new cryo-EM suite in the Centre for Advanced Microscopy (CAM) at ANU and the installation of the JEOL developed MicroED data acquisition software, RecorderTM in July 2022, it became my responsibility to make this technique available to the local community. Having never worked with protein crystallography and with little experience in 3D diffraction data collection (MicroED) and data processing, it quickly became obvious that the shadowing program was a great opportunity to fast-track my knowledge.
Before I went for training, I worked on amassing experience with test samples to get a fundamental knowledge about the technique. This was when I attained real appreciation for the technique’s potential benefits to the Microscopy Australia user base.
Since the 90’s, 3D electron diffraction data collection routines have been established by several groups, which led to the adoption of several names such as automated diffraction tomography (ADT), continuous rotation electron diffraction (cRED) and electron diffraction tomography (EDT) to list a few. For more details on these historical developments the reader should refer to Gruene et. al. and references within (Gruene, 2021). However, it was not until 2013 that the term micro electron diffraction (MicroED) came into light with the experiments led by Tamir Gonen on lysozyme microcrystals (Shi, 2013), bringing electron diffraction into the vastly important field of protein crystallography. Owing to many advances in cryo-EM made in the past decade, including better detectors, more stable stages, and software, the technique has seen continuous development and it is now available in several laboratories* across Australia.
To group all these different acronyms that describe essentially the same technique, Gemmi et. al. (Gemmi, 2019) have created the simple unifying term 3D ED, which I will refer to from now on for simplicity. The 3D ED technique consists of a transmission electron microscopy experiment where the sample is rotated continuously under the electron beam while a fast detector records the obtained diffraction signal. Given that the experiment is an exact analogue of the well-established x-ray diffraction (XRD) experiments used in Structural Biology and Chemical Crystallography we are able to make use of most of the software analysis tools developed in these fields. Despite its similarity there are a few aspects that set 3D ED apart from its x-ray counterpart. Firstly, due to its stronger interaction with matter, electrons offer the examination of protein crystals orders of magnitude smaller than required for conventional XRD. Hence, 3D ED offers a great opportunity to investigate particularly difficult targets for x-ray crystallography, such as membrane proteins and peptides whose pathway from purification to crystallization of larger, well diffracting crystals would be cost prohibitive (Hattne, 2015 and Xu, 2019). In addition to investigating smaller protein crystals, it has recently been demonstrated that electron diffraction is suitable to study small molecule complexes such as pharmaceutical complexes (Jones, 2018). Finally, x-rays scatter mostly from the electron cloud. Therefore, structures obtained from XRD patterns display a 3D representation of the sample’s electron densities. For this reason, Hydrogen atoms are generally hard to resolve with XRD since it carries only a single electron (Palatinus, 2017). In electron diffraction the scattering is much stronger from both the electrons and the atomic nucleus. This allows for direct observation of hydrogen bonds even when using lower resolution data. Convinced of its usefulness, I needed a contact to access 3D ED training.
I was lucky to be introduced to Dr. Hongyi Xu from the Department of Materials and Environmental Chemistry (MMK) at Stockholm University by our colleagues at the Centre for Microscopy and Microanalysis in the University of Queensland, Dr. Na’ama Koifman and Dr. Kasun Athukorala. Not only was Dr. Xu very generous with his time helping me analyze and solve my first structure with 3D ED, but he also invited me to their lab in Sweden and witness their operations at MMK. Fortunately, the invitation was met with the support from CAM’s director Dr. Melanie Rug who approved my training. While visiting Dr. Xu I had the chance to collect electron diffraction and screen the data using the locally developed software Instamatic (Cichoka, 2018 and Smeets, 2018) and REDp (Wan, 2013 and Zhang, 2010). Dr. Xu and his team** also shared with me several of their newly developed sample preparation and data acquisition protocols, some of which are yet to be published.
Upon returning to Australia, I have been working to put into practice what I’ve learned as well as working to establish some of the new protocols I observed in Sweden, especially regarding data acquisition routines with Instamatic. I feel very grateful to Microscopy Australia and CAM for the support and to the team at MMK for sharing their valuable time and resources with me. In conclusion, the experience in Sweden not only provided me with new skills and experiences but also gave me great confidence in the future of 3D ED in supporting structural characterization of small molecules, peptides, and protein crystals otherwise un-accessible by XRD.
[1] (Cichocka, 2018) M.O. Cichocka, J. Ångström, B. Wang, X. Zou, and S. Smeets. J. Appl. Cryst., 51, 1652–1661, 2018.
[2] (Gemmi, 2019) M. Gemmi, E. Mugnaioli, T. E. Gorelik, U. Kolb, L. Palantinus, P. Boullay, S. Hovmöller, and J. P. Abrahams. ACS Cent. Sci. 5, 1315-1329, 2019.
[3] (Gruene, 2021) T. Gruene, J. J. Holstein, G. H. Clever, and Keppler. Nature Reviews Chemistry 5, 660-668, 2021.
[4] (Hattne, 2015) J. Hattne, F. E. Reyes, B. L. Nannenga, D. Shi, M. J. de La Cruz, A. G. W. Leslie, and T. Gonen. Acta Cryst. A71, 353-360, 2015.
[5] (Jones, 2018) C. Jones, M. W. Martynowycz, J. Hattne, T. J. Fulton, B. M. Stoltz, J. A. Rodriguez, H. M. Nelson, T. Gonen. ACS Cent. Sci. 4(11), 1587-1592, 2018.
[6] (Palatinus, 2019) L. Palatinus, P. Brázda, O. Perez, M. Klementova, S. Petit, V. Eigner, M. Zaarour, and S. Mintova. Science 13, 166-159, 2017.
[7] (Shi, 2013) D. Shi, B. L. Nannnenga, M. G. Iadanza, T. Gonen. eLife, 2013.
[8] (Smeets, 2018) S. Smeets, X. Zou, and W. Wan. J. Appl. Cryst., 51, 1262–1273, 2018.
[9] (Wan, 2013) Wan, W.; Sun, J.; Su, J.; Hovmöller, S.; Zou, X., Appl. Crystallogr., 46, 1863– 1873, 2013.
[10] (Xu, 2019) H. Xu, H. Lebrette, M. T. Clabbers, J. Zhao, J. J. Griese, X. Zou, and M. Högbom. Sci. Adv. 5(8), eaax4621, 2019.
[11] (Zhang, 2010) Zhang, D.; Oleynikov, P.; Hovmoller, S.; Zou, X., Z. Kristallogr. Cryst. Mater., 225, 94-102, 2010.
Dr. Kasun S. Athukorala Arachchige, Centre for Microscopy and Microanalysis, University of Queensland.
Dr. James Bouwer, Molecular Horizons, University of Wollongong.
Assoc./Prof. Martin Saunders, Centre for Microscopy, Characterization & Analysis, University of Western Australia.
Dr. Ashley Slattery, Adelaide Microscopy, University of Adelaide.
Dr. Hari Venugopal, Ramaciotti Centre for Cryo-Electron Microscopy, Monash University.
**Thank you very much to the team at MMK. Dr Hongyi Xu, Prof. Xiaodong Zou, Ms Laura C. Pacoste, Dr Gerhardt Hofer, Mr Lei Wang, Dr Taimin Yang and Ms Jiaoyan Xu
October 5, 2023
