Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography
To overcome the inherent low-throughput characteristic of CET data collection, improve the resolution of SVA and extend its application to a wider set of samples including low molecular weight targets, here, we: (1) use beam-image shift navigation to multiply the number of regions of interest imaged at each mechanical step, (2) integrate image processing into data collection to compensate the imprecision of the stage using geometrical constraints of multiplexed ROIs, and (3) introduce improved data processing routines for high-resolution structure determination using constrained single particle tomography (CSPT). As proof of concept, we collected tilt-series using beam image-shift electron cryo-tomography (BISECT) and determined the 3.6 Å resolution structure of a low molecular weight target (~300kDa).
| 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. |
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