CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples
- 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
Tilt estimation and correction in CTFFIND5.
(a) Power spectra are calculated in 128×128 pixel patches as indicated on a representative micrograph. The dots represent the locations of the patches and the box indicates patch size. (b) A model …
Figure 2
Validation of tilt estimation using tilt series data and comparison of defocus estimation using CTFFIND4 and CTFFIND5.
(a) Estimated tilt angle and axis of 40 micrographs of a tilt series taken on a focused ion beam (FIB)-milled biological specimen. For each image the tilt angle (dots, top plot) and tilt axis …
Figure 3
Sample thickness estimation by fitting Thon ring patterns.
(a) Comparison of the contrast transfer function (CTF) model used in CTFFIND4, and after applying the modulation function (right) described by McMullan et al., 2015. A star symbol (*) denotes the …
Figure 4
Validation of sample thickness estimation in CTFFIND5 by comparing the estimates to the intensity attenuation by the zero-loss energy filter.
An estimation of the linear relationship using the RANSAC algorithm results in a slope of 1/316.6 nm and an x-axis intercept at –14 nm (red dashed line). Data points that were labeled as outliers by …
Figure 5
Validation of sample thickness estimation in CTFFIND5 by tomography.
The distribution of thickness measurements in seven tomograms are shown as box plots with the median indicated by a red line. For each tomogram, the thickness was measured in three different places. …
Figure 6
Contrast transfer function (CTF) correction of medium-magnification overviews.
(a) Representative area of a micrograph of a cellular sample at a pixel size of 40 Å without CTF correction. (b) Fit of the power spectrum of the micrograph shown in panel (a) CTF model. (c–d) The …
Tables
Table 1
Comparison of CTFFIND5 estimation of sample tilt with crystallographic analysis.
| Image | Axis angle φ | Tilt angle θ | |||||
|---|---|---|---|---|---|---|---|
| Crystallog. | CTFFIND5 | ∆φ | Crystallog. | CTFFIND5 | ∆θ | ||
| 530394 | 93.28 | 94.98 | –1.7 | 19.6 | 20.69 | –1.09 | |
| 530419 | 109.78 | 106.51 | 3.27 | 18.66 | 16.04 | 2.62 | |
| 530430 | 104.38 | 101.13 | 3.25 | 21.32 | 20.37 | 0.95 | |
| 530444 | 98.39 | 97.62 | 0.77 | 20.72 | 20.88 | –0.16 | |
| 660027 | 99.68 | 102.34 | –2.66 | 19.4 | 22.39 | –2.99 | |
| 540149 | 94.45 | 85.84 | 8.61 | 43.08 | 44.59 | –1.51 | |
| 540291 | 96.16 | 98.1 | –1.94 | 45.11 | 40.68 | 4.43 | |
| 540302 | 93.98 | 93.39 | 0.59 | 44.7 | 44.21 | 0.49 | |
| 540313 | 95.34 | 95.13 | 0.21 | 44.03 | 46.49 | –2.46 | |
| 660183 | 97.69 | 97.27 | 0.42 | 48.13 | 48.99 | –0.86 | |
| 550069 | 90.08 | 92.55 | –2.47 | 60.46 | 60.83 | –0.37 | |
| 550089 | 91.48 | 92.04 | –0.56 | 60.5 | 60.72 | –0.22 | |
| 660291 | 93.23 | 92.19 | 1.04 | 57.59 | 59.19 | –1.60 | |
| 660421 | 89.32 | 89.06 | 0.26 | 61.36 | 60.01 | 1.35 | |
| 680341 | 89.67 | 90.02 | –0.35 | 58.68 | 59.62 | –0.94 | |
| 530345 | N/A | 108.6 | 0 | 0.84 | –0.84 | ||
| 530356 | N/A | 231.17 | 0 | 1.93 | –1.93 | ||
| 530358 | N/A | 56.58 | 0 | 1.29 | –1.29 | ||
| 530375 | N/A | 3.21 | 0 | 0.79 | –0.79 | ||
| 530378 | N/A | 67.6 | 0 | 2.17 | –2.17 | ||
Table 2
Runtime of CTFFIND5 on representative micrographs.
| Micrograph | 1 | 2 | 3 | ||
|---|---|---|---|---|---|
| Image properties | |||||
| Image size | 4070×2892 | 2880×2046 | 4746×3370 | ||
| Pixel size (Å) | 1.5 | 4.175 | 2.5 | ||
| Runtime (s) | |||||
| Tilt | Thickness | ||||
| – | – | 0.9±0.1 | 0.7±0.1 | 1.7±0.1 | |
| + | – | 39.0±0.2 | 208±1 | 173.4±0.1 | |
| – | + | 1.4±0.1 | 1.3±0.1 | 2.4±0.1 | |
| + | + | 39.5±0.1 | 209±1 | 173.0±0.1 | |
