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| Item Type: | Article |
|---|---|
| Title: | Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM |
| Creators Name: | Sanchez, R.M., Zhang, Y., Chen, W., Dietrich, L. and Kudryashev, M. |
| Abstract: | Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution. |
| Keywords: | Calcium Channels, Cryoelectron Microscopy, Electron Microscope Tomography, Computer-Assisted Image Processing, Macromolecular Substances, Ryanodine Receptor Calcium Release Channel, Signal-To-Noise Ratio, Single Molecule Imaging, Workflow |
| Source: | Nature Communications |
| ISSN: | 2041-1723 |
| Publisher: | Nature Publishing Group |
| Volume: | 11 |
| Number: | 1 |
| Page Range: | 3709 |
| Date: | 24 July 2020 |
| Official Publication: | https://doi.org/10.1038/s41467-020-17466-0 |
| PubMed: | View item in PubMed |
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