Unbend, correction of local beam-induced sample motion in cryo-EM images using a 3D spline model
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, United States
- Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, United States
Figures
Figure 1
Uniform cubic spline curve.
(a) Diagram showing the core components of a cubic B-spline curve, such as knots, control points, and the uniform knot positions . (b) Diagram showing the free-end boundary condition for a cubic B-spline.
Figure 2
3D spline model knot grid construction and shift field generation.
(a) Diagram showing the position of knots along the exposure accumulation direction and the shift amount along x for each movie frame. (b) A 3D diagram showing the cubic B-spline along the exposure accumulation direction, with red arrows representing the shifts where the knots are located (black dots on the gray line), and the cyan curve showing the spline curve for interpolating the shifts for each frame. (c, d) Shift field along x and y showing the shift amount of each pixel obtained from the bicubic B-splines on 30×30 pixels frame (for demonstration only). (e) Schematic image grid before (gray grids) and after (cyan grid) applying the shift fields in (c, d). The red arrows represent the shift of patches. Black dots represent knot positions.
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Figure 2—source data 1
Unbend movie alignment and local motion correction algorithm.
- https://cdn.elifesciences.org/articles/109119/elife-109119-fig2-data1-v1.xlsx
Figure 3
A micrograph of whole-cell M. pneumoniae.
(a) Motion trajectory panel in the cisTEM graphical user interface (GUI). (b) The cropped area shown in (a) without local distortion correction, experiencing blurring due to the local distortion (upper) and the power spectrum (lower). (c) The cropped area (upper) and the power spectrum of the local distortion-corrected micrograph (lower). (d) Detected particles (white) using 2D template matching (2DTM) on the full-frame aligned micrograph. (e) Detected particles (white) using 2DTM on the local-distortion corrected micrograph.
Figure 4 with 5 supplements
Patch shift in different types of samples.
(a) Box plots of the standard deviation of patch motions calculated for micrographs of different types of samples. (b) The mean patch shifts for each micrograph, colored by sample type.
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Figure 4—source data 1
Summary statistics of per-micrograph mean patch shifts by sample type.
- https://cdn.elifesciences.org/articles/109119/elife-109119-fig4-data1-v1.xlsx
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Figure 4—source data 2
Motion and equivalent strain summary table for micrographs shown in Figure 4—figure supplements 1–5.
- https://cdn.elifesciences.org/articles/109119/elife-109119-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
M. pneumoniae sample.
(a) Screenshot of the cisTEM graphical user interface (GUI) displaying the motion-corrected micrograph and patch trajectories. (b) Micrograph based on full-frame alignment. (c) Von Mises equivalent strain field. (d) Total equivalent strain field.
Figure 4—figure supplement 2
BS-C-1 cell edge sample.
(a) Screenshot of the cisTEM graphical user interface (GUI) displaying the motion-corrected micrograph and patch trajectories. (b) Micrograph based on full-frame alignment. (c) Von Mises equivalent strain field. (d) Total equivalent strain field.
Figure 4—figure supplement 3
M. musculus ER-HoxB8 cell lamella sample.
(a) Screenshot of the cisTEM graphical user interface (GUI) displaying the motion-corrected micrograph and patch trajectories. (b) Micrograph based on full-frame alignment. (c) Von Mises equivalent strain field. (d) Total equivalent strain field.
Figure 4—figure supplement 4
S. cerevisiae lamella sample.
(a) Screenshot of the cisTEM graphical user interface (GUI) displaying the motion-corrected micrograph and patch trajectories. (b) Micrograph based on full-frame alignment. (c) Von Mises equivalent strain field. (d) Total equivalent strain field.
Figure 4—figure supplement 5
BS-C-1 cell lysate sample.
(a) Screenshot of the cisTEM graphical user interface (GUI) displaying the motion-corrected micrograph and patch trajectories. (b) Micrograph based on full-frame alignment. (c) Von Mises equivalent strain field. (d) Total equivalent strain field.
Figure 5
Magnitude of deformation correction of the micrograph shown in Figure 3a.
(a) Von Mises equivalent strain and (b) total equivalent strain.
Figure 6
Evaluation of local motion correction using Unbend across different sample types.
(a) Box plots displaying the distribution of the number of 2D template matching (2DTM) detections per micrograph. (b) Histograms of the 2DTM signal-to-noise ratio (SNR) of detected 2DTM targets. Unbend improves the overall distribution of SNR values for all sample types. (c) 2DTM SNR difference between Unbend and Unblur. Across all sample types, Unbend led to improvements in either the number of detected targets or 2DTM SNR values, or both.
Figure 7
Impact of patch-based motion correction on the detection of 60S subunits in focused ion beam-milled lamellae analyzed in a large dataset.
(a) Histogram showing the percentage of increase in 60S detection in montages processed using Unbend, compared to Unblur. (b) Scatterplot of the number of detected 60S per exposure in a representative montage that did not show a substantial increase of detection after processing with Unbend, compared to Unblur. (c) Similar to panel (b), but for a representative montage with a more substantial increase of detected targets after processing with Unbend, compared to Unblur.
Figure 8
Comparison of local motion correction methods.
(a) Box plots showing the distribution of the number of 2D template matching (2DTM) detections per micrograph for each motion correction method. (b) Distribution of 2DTM signal-to-noise ratio (SNR) differences between Unbend and the other tested methods for particles detected in common (). Vertical lines mark the zero-crossing in the sorted distribution for each method. Percentages in the legend indicate the fraction of commonly detected particles with higher SNR in Unbend-processed micrographs.
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Figure 8—source data 1
Statistics table for detected particles from micrographs processed by different software.
- https://cdn.elifesciences.org/articles/109119/elife-109119-fig8-data1-v1.xlsx
Tables
Table 1
Unbend runtimes on movies analyzed in Figure 6.
| M. pneumoniae | Aethiops: BS-C-1(cell edge) | S. cerevisiae | Musculus:ER-HoxB8 | Aethiops: BS-C-1(cell lysate) | |
|---|---|---|---|---|---|
| Input movie size (pixels) | 5760×4092 | 5760×4092 | 11520×8184 | 11520×8184 | 11520×8184 |
| No. of frames per movie | 24 | 30 | 50 | 75 | 30 |
| Exposure per frame (e–/Å2) | 1.296 | 1.021 | 0.600 | 0.600 | 1.004 |
| No. of movies | 64 | 65 | 30 | 59 | 76 |
| Input pixel size (Å/pixel) | 1.053 | 1.33 | 0.53 | 0.415 | 0.415 |
| Defocus (µm) (mean ± standard deviation) | 1.3±0.5 | 1.5±0.3 | 0.4±0.1 | 1.0±0.5 | 1.2±0.4 |
| Patch number | 8×6 | 10×8 | 8×6 | 7×5 | 7×5 |
| Output image size (pixels) | 4044×2873 | 5107×3628 | 4070×2892 | 3187×2264 | 3187×2264 |
| Output pixel size (Å/pixel) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| Runtime per movie (mean ± standard deviation) | 1m34s±5 s | 3m21s±35 s | 7m58s±26 s | 12m30s±43 s | 4m49s±9 s |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (M. pneumonia) | M129 | O’Reilly et al., 2020; Lucas et al., 2021 | ATCC 29342 | |
| Cell line (C. aethiops) | BS-C-1 | ATCC | CCL-26; RRIDs:CVCL_0607 | |
| Cell line (M. musculus) | ER-HoxB8 | Elferich et al., 2022 | ||
| Cell line (S. cerevisiae) | BY4741 | Lucas et al., 2022 | ATCC S288C | |
| Cell line (C. albicans) | PY6413 | Serrano et al., 2026 | ||
| Software, algorithm | cisTEM | Lucas et al., 2021; Grant et al., 2018; Elferich et al., 2024 | https://cistem.org/ |
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Unbend, correction of local beam-induced sample motion in cryo-EM images using a 3D spline model
eLife 14:RP109119.
https://doi.org/10.7554/eLife.109119.3
