Article: Single particle cryo-electron microscopy with an enhanced 200 kV cryo-TEM configuration achieves near-atomic resolution.
bioRxiv : the preprint server for biology
2024
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 ... ...
| 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 (Å) 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 Å 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 Å, 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 Å reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg |
|---|---|
| Language | English |
| Publishing date | 2024-05-10 |
| Publishing country | United States |
| Document type | Preprint |
| DOI | 10.1101/2024.05.07.593029 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
More links
Kategorien
Inter-library loan at ZB MED
Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.