Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

Abstract

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.

Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

“…Our data (Supplementary Fig. 5d) support a common observation that rotation of the inplane image shift deviates significantly from the designated coordinates as the stage tilt increases 23 . This departure in image shift values introduced by global tile rotation was much larger than the applied translation alone at lower tilts and became particularly irregular as the stage was tilted to degrees greater than ±30°.…”

“…9c). We used CTFFIND4 for tilted defocus determination 23 . CTFFIND4 reported the high-resolution limit of detected Thon rings that ranged from 8 to 20 Å for individual tiles and stitched montages at 0° tilt and high tilts (Supplementary Fig.…”

Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces

“…With these requirements, dispersed molecular complexes fare better, because blotting parameters can be adjusted to create ice as thin as possible. In vitro samples like this have achieved sub 5Å resolution, either as part of assemblies [32,33] or as single copies [34,35] (see Figure 3). The latter is as close to the resolution of the same (~300 kDa) particle as solved with SPA.…”

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

“…In recent years, substantial developments in cryo-ET data acquisition and STA processing have resulted in high-resolution structures of challenging samples that are not accessible by any other method (Bäuerlein and Baumeister, 2021). However, while highresolutions at ~4Å can now be reached for some specimens, such as ribosomes (O'Reilly et al, 2020;Tegunov et al, 2021), selected virus assemblies (Dick et al, 2020;Schur et al, 2016), and apoferritin or dNTPase in vitro (Bouvette et al, 2021;Tegunov et al, 2021), the average resolution of cryo-ET-derived structures in the electron microscopy databank (EMDB) has only marginally increased over the last few years (with an average resolution of ~21 Å in 2021) and has remained behind what is now commonly achieved in single-particle particle (SPA) cryo-EM (~5.3 Å average resolution in 2021).…”

“…Of the above-mentioned limitations, the last point has recently been addressed in several developments that exploit efficient data acquisition methods, more stable stages and faster cameras (Bouvette et al, 2021;Chreifi et al, 2019;Eisenstein et al, 2019). This has allowed obtaining datasets of hundreds of tomograms within a short time.…”