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Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.
- Source :
-
BioRxiv : the preprint server for biology [bioRxiv] 2023 Jul 15. Date of Electronic Publication: 2023 Jul 15. - Publication Year :
- 2023
-
Abstract
- Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.
Details
- Language :
- English
- Database :
- MEDLINE
- Journal :
- BioRxiv : the preprint server for biology
- Accession number :
- 37503021
- Full Text :
- https://doi.org/10.1101/2023.07.14.548955