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bioRxiv · 10.1101/2024.05.07.593029

Single particle cryo-electron microscopy with an enhanced 200 kV cryo-TEM configuration achieves near-atomic resolution

Jia, L.·Ruben, E. E.·Suarez, H. J.·Olsen, S. K.·Wasmuth, E. V.
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Abstract

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom ([A]) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 [A] and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 [A], respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset ([≤]1,000 micrographs) was sufficient to generate a 3.3 [A] reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg+2. In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.

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Jia, L., Ruben, E. E., Suarez, H. J., Olsen, S. K., Wasmuth, E. V.. 2024-05-10. Single particle cryo-electron microscopy with an enhanced 200 kV cryo-TEM configuration achieves near-atomic resolution. https://doi.org/10.1101/2024.05.07.593029

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