Computational Tools to Improve Visualization by Cryo-Electron Tomography

Abstract

Cryo-electron tomography (CryoET) can directly visualize frozen, hydrated macromolecules inside cells without staining artifacts or fluorescent tags at nanometer resolution. However, imaging tilted specimens is limited to ±60° tilt, causing a “missing wedge” of data that induces smearing artifacts in tomograms. Furthermore, each image is subject to a defocusing artifact known as the contrast transfer function (CTF) of the electron microscope. Lastly, high noise and low contrast make feature identification difficult and error prone. Although contrast is typically improved by denoising (filtering), the tomographic reconstruction process introduces additional artifacts that can be accentuated when filters are applied to tomograms in 3-D.We developed tools for CTF correction in cryoET tiltseries and to fill in the missing wedge. We implemented them in EMAN2, a widely used software package for cryo elctron microscopy data processing. Using our first too, we applied CTF correction on cryoET tiltseries of viral particles of known structure recorded with a direct electron detector (DE20). Images collected in “movie-mode” were first corrected for drift. We achieve a resolution improvement from ∼25A to ∼13A. Our second tool “fills in” the missing wedge with reprojections of tomographic data from angular directions not accessible experimentally in the electron microscope. Results on viral particle data show decreased missing wedge artifacts (smearing). We also applied pre-reconstruction denoising filters directly to 2-D images in tiltseries, which improved the accuracy of automated particle picking (measured against manual particle picking, considered ground truth).Lastly, we applied pre-reconstruction denoising filters to tiltseries of mammalian cells to minimize artifacts and enhance the reliability of feature identification. For example, in platelets, microtubules and the cell membrane become clearer with pre-filtration, compared to the standard post-reconstruction filtering approach, and mitochondria become more easily distinguishable from granules.