Current data processing strategies for cryo-electron tomography and subtomogram averaging

Abstract

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

Microscopic Visualization of Cell-Cell Adhesion Complexes at Micro and Nanoscale

“…This can get very complex, time- and computer-demanding depending on the technique used. 3D acquired data (from ET, SXT and volume-SEM) needs some post-processing, including corrections, stack alignments and sub-tomogram averaging, before analysis can be performed ( Schur 2019 ; Pyle and Zanetti, 2021 ). EM and SXT data analysis is often carried out by specialists with experience in the identification and interpretation of biological features in the complex grayscale world of this type of imaging.…”

Structure Determination of Microtubules and Pili: Past, Present, and Future Directions

“…The ability to faithfully localise and identify macromolecules on MTs in situ is likely to be a prominent target in the future direction of the field. With the advent of a revolution in macromolecular structure prediction (Jumper et al, 2021) combined with expanding dataset sizes and increasing subtomogram averaging resolutions due to steady improvements in cryo-ET sample preparation (FIB), data-collection and image processing (Figure 1) (Turk and Baumeister, 2020;Hylton and Swulius, 2021;Pyle and Zanetti, 2021), this goal is looking more and more plausible.…”

“…Furthermore, a significant amount of sample heterogeneity can also now be tolerated and even utilised, with snapshots of different conformational states and multimeric arrangements revealing the dynamics of macromolecular machines. Whilst remaining more challenging than single-particle cryo-EM, in the last 5 years or so cryo-electron tomography (cryo-ET) with sub-tomogram averaging has become more capable of achieving sub-nanometer resolutions in situ and near-atomic resolutions with purified macromolecular preparations (Schur, 2019;Turk and Baumeister, 2020;Pyle and Zanetti, 2021).…”

Improving a Rapid Alignment Method of Tomography Projections by a Parallel Approach

“…Marker-based alignment methods are a common solution to the problem, especially in the case of biological samples which exhibit low absorption contrast and/or not welldefined features; at the cost of decreasing the sample visibility (this is the measure of how critical this step is), gold nano-beads are added to the sample solutions before cryo-fixation [2] and are tracked in a post-acquisition procedure among each projection. IMOD [18][19][20] and many other software frameworks [14,[21][22][23][24][25][26] reviewed in, e.g., [27] are used to manage this delicate pre-processing step. However, this approach is questionable, as for many biological specimens, it is extremely difficult to control the spatial distribution of such fiducials.…”