High-resolution cryo-EM at 200kV enabled by Selectris-X and Falcon 4

Session

Poster Session 4

Authors

Dr Adrian Koh (1), Dr Dimple Karia (1), Dr Sebastian Unger (1), Mr Dennis Cats (1), Mr Erwin de Jong (1), Dr Lingbo Yu (1), Dr Abhay Kotecha (1)

Affiliations

1. Thermo Fisher Scientific

Keywords

Glacios, Selectris, Falcon 4, 200kV, High-resolution, Single Particle Analysis, Energy Filter 

Abstract text

In the recent years, electron-cryo microscopy (cryo‐EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules[1,2]. While state-of-the-art 300 kV Cryo-TEM has contributed to most of the high-resolution SPA structures to date, we believe that 200 kV systems with energy filters can deliver similar results. Here, we show data from the 200 kV Glacios cryo-transmission electron microscope (Cryo-TEM) equipped with the Selectris-X energy filter and Falcon 4 detector resulting in high resolution maps sufficient for de novo atomic model building for soluble as well as membrane proteins. 

 

While the latest “resolution revolution” allows scientists to visualize structural detail down to individual hydrogen atoms, previously believed to be impossible with electron microscopy, these gains have largely been limited to well-off institutions able to afford the costs of purchasing, hosting, and maintaining the 300 kV Cryo-TEMs. As early as 2014 [3] a 4 Å resolution SPA reconstruction was reported using data collected on 200kV cryo-TEMs. But it was not until 2020 that the 2 Å barrier was breached by Wu et al. [4] presenting a 1.75 Å density map of Apoferritin from a 200 kV Talos Arctica. While these record-breaking events show the potential of the instrument, the actual number of high-resolution depositions in EMDB from 200 kV microscopes are far overshadowed by 300 kV microscopes. To date, there are only 59 SPA entries in EMDB that have a reported resolution higher than 3 Å that were imaged on 200kV systems compared to 1022 SPA entries from 300kV systems. Thus, we set out to investigate if a 200 kV Glacios with a Selectris-X energy filter can produce data leading to sub-3 Å resolution structures. 

 

All data collection was carried out on a 200 kV Glacios Cryo-TEM with a Falcon 4 mounted at the end of the Selectris-X energy filter. Datasets were collected with and without insertion of the energy slit on the same grid with similar ice thickness. We were very encouraged by the 1.9 Å resolution for ApoF, 2.3 Å for T20S Proteosome, and 2.8 Å for homo-pentameric human GABAA receptor without energy filtration. However, when data was collected with a 10 eV energy slit, it resulted in significantly improved resolutions across the board, resulting in 1.7 Å resolution for ApoF, 2.1 Å for T20S Proteosome, and 2.4 Å for homo-pentameric human GABAA receptor. To push the setup further, we decided to compared data collected on a 300 kV Krios with a BioQuantum-K3. GLP‐1R (glucagon‐like peptide‐1 receptor), a validated target for the treatment of type 2 diabetes and obesity, was recently solved to 2.1 Å with data collected on a Krios equipped with a BioQuantum-K3 [5]. Data collected on the Glacios with Selectris-X on the same sample yielded a 2.6 Å density map GLP‐1R. These results clearly demonstrate the potential utility of a Selectris-X, Falcon 4 equipped 200 kV Glacios Cryo-TEM for high-resolution SPA experiments.

References

[1] Li, X., Mooney, P., Zheng, S., Booth, C.R., Braunfeld, M.B., Gubbens, S., Agard, D.A. and Cheng, Y., 2013. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature methods, 10(6), pp.584-590. 

[2] Bai, X.C., 2021. Seeing Atoms by Single-Particle Cryo-EM. Trends in biochemical sciences. 

[3] Campbell MG, Kearney BM, Cheng A, Potter CS, Johnson JE, Carragher B, Veesler D. Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV. J Struct Biol. 2014 Nov;188(2) 183-187. doi:10.1016/j.jsb.2014.09.008. PMID: 25278130; PMCID: PMC4497823.

[4] Wu M, Lander GC, Herzik MA Jr. Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV. Journal of Structural Biology: X. 2020 ;4:100020. DOI: 10.1016/j.yjsbx.2020.100020. 

[5] Zhang, X., Belousoff, M.J., Zhao, P., Kooistra, A.J., Truong, T.T., Ang, S.Y., Underwood, C.R., Egebjerg, T., Šenel, P., Stewart, G.D. and Liang, Y.L., 2020. Differential GLP-1R binding and activation by peptide and non-peptide agonists. Molecular Cell, 80(3), pp.485-500.