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 are simultaneously consistent with high-throughput and high-resolution imaging. 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 (~300kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.

Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data

“…Within this manuscript we did not intend to reveal new biological insights into the actin network architecture, but rather showcase the functionalities of the introduced toolbox. We expect that with increased throughput in data acquisition ( 4 – 6 ), large datasets for a variety of samples can be acquired in short time, further highlighting the importance to develop ease-of-use tools allowing efficient analysis of the wealth of biological data contained within cellular cryo-electron tomograms. Indeed, we believe that the real power of the toolbox comes with the time-efficient analysis of large datasets, and will allow the detection of subtle ultrastructural differences between experimental conditions, e.g., when comparing multiple genetic knockout clones with mild phenotypes.…”

“…Hence, while the potential of cryo-ET as a qualitative method is commonly accepted for applications where the analysis of a few tomograms is sufficient to detect and describe novel subcellular features, its potential as a quantitative technique to compare subtle differences among genetically distinct samples is not yet fully realized. Recent improvements in cryo-EM sample preparation (3), automated EM data acquisition (4)(5)(6), image processing workflows (7), and data analysis allow the evaluation of large datasets and comparison of various in situ features between multiple experimental conditions. These improvements, although very suitable for being combined with the nowadays relatively straightforward genetic manipulation of cell lines via CRISPR/Cas9 techniques, are yet to be routinely applied in workflows that facilitate the highthroughput analysis and comparison of ultrastructural characteristics between genetically modified cell lines.…”

“…Recent improvements in cryo-EM sample preparation ( 3 ), automated EM data acquisition ( 4 – 6 ), image processing workflows ( 7 ), and data analysis allow the evaluation of large datasets and comparison of various in situ features between multiple experimental conditions. These improvements, although very suitable for being combined with the nowadays relatively straightforward genetic manipulation of cell lines via CRISPR/Cas9 techniques, are yet to be routinely applied in workflows that facilitate the high-throughput analysis and comparison of ultrastructural characteristics between genetically modified cell lines.…”

Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

“…Here we present our application of micropatterning to extend this technique for cryo-EM studies of multiple cell types by utilizing features such as high resolution and contactless patterning of the PRIMO system to pattern TEM grids. Modern, advanced electron microscopes and software packages now support streamlined automated cryo-EM and cryo-ET data collection where hundreds to thousands of positions can be targeted and imaged within a few days [25][26][27][28] . One significant limiting factor for whole-cell cryo-ET workflows has been obtaining sufficient numbers of collectable targets per grid.…”

“…Modern, advanced electron microscopes and software packages now support streamlined automated cryo-EM and cryo-ET data collection where hundreds to thousands of positions can be targeted and imaged within a few days 25-28 . One significant limiting factor for whole-cell cryo-ET workflows has been obtaining sufficient numbers of collectable targets per grid.…”