Figures and data in CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples | eLife

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6 figures, 2 tables and 1 additional file

Figures

Figure 1

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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 of the expected power spectrum in each patch given an average defocus $Δ f$ , tilt angle $θ$ , and tilt axis $ϕ$ is compared to the actual power spectra of tiles. After an optimal set of $θ$ and $ϕ$ has been found a corrected power spectrum is calculated by summing the tile power spectra, scaled to correct for the defocus difference. Power spectra shown are an exaggerated example. The convention of $ϕ$ as a counterclockwise rotation from the x-axis is indicated. (c) Comparison of the original power spectrum (EPA, solid line, blue) to the tilt-corrected power spectrum (solid line, black). The tilt-corrected power spectrum exhibits clear peaks at higher spatial resolution than the uncorrected power spectrum, as evident by the 'goodness-of-fit' scores (dashed lines). The estimated contrast transfer function (CTF) parameters are $Δ f_{1} = 10603 Å, Δ f_{2} = 10193 Å, α = {85.9}^{\circ}$ for the uncorrected power spectrum and $Δ f_{1} = 10492 Å, Δ f_{2} = 10342 Å, α = {81.2}^{\circ}, θ = {12.3}^{\circ}, ϕ = {261.6}^{\circ}$ for the tilt-corrected power spectrum. The fit resolution is 5.9 Å for the uncorrected power spectrum (dashed line, blue) and 4.6 Å for the tilt-corrected spectrum (dashed line, black).

Figure 2

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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 direction (crosses, second plot) are plotted as a function of the nominal stage angle. The data were fitted to a model of the specimen tilt and constant stage tilt axis before tilting the stage (red dashed line in first and second plot). The estimated stage tilt axis has an angle of 178.2° and the estimated specimen pre-tilt is 20.6° with a tilt axis of 171.8°, which is consistent with the FIB-milling angle of 20° and manual alignment of the milling direction to the goniometer tilt axis. The third plot shows the fit residuals for tilt angle and axis are plotted. The fourth and fifth plots show a plot of the estimated defocus value and fit resolution for each tilt image, as derived from CTFFIND4 (red) or CTFFIND5 (black). (b) Data for another tilt series plotted as described for (a). The estimated stage tilt axis is 179.8°, the estimated specimen pre-tilt is –21.9° with a tilt axis of 183.8°. This is consistent with this grid being inserted in the opposite orientation as the grid shown in (a), but still with a rough alignment of milling direction and tilt axis.

Figure 3

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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 position of the first zero in the CTF and a pound symbol (#) denotes the position of the first node. (b–d) Representative examples of Thon ring fitting in micrographs without (top-left graph) and with (bottom-left graph) thickness estimation. The micrograph is shown to the right. Each graph shows that equiphase averaging (EPA) of the power spectrum in solid black lines, the fitted CTF model in dashed red lines, and the goodness of fit indicator as a dotted blue lines. (b) Thon ring fitting of a micrograph taken from a focused ion beam (FIB) milling-derived lamella. The tilt of the specimen was estimated to be 12.3°. When fitting without thickness estimation the estimated parameters were $Δ f_{1} = 10492 Å, Δ f_{2} = 10342 Å, α = {81.2}^{\circ}$ . When taking sample thickness into account the estimated parameters were $Δ f_{1} = 10481 Å, Δ f_{2} = 10286 Å, α = {69.6}^{\circ}, t = 969 Å$ . The estimated fit resolution was $4.6 Å$ and $3.4 Å$ without and with sample thickness estimation, respectively. (c) Thon ring fitting of a micrograph taken from a FIB milling-derived lamella. The tilt of the specimen was estimated to be 6.7°. When fitting without thickness estimation the estimated parameters were $Δ f_{1} = 8002 Å, Δ f_{2} = 7717 Å, α = {73.4}^{\circ}$ . When taking sample thickness into account the estimated parameters were $Δ f_{1} = 8549 Å, Δ f_{2} = 8343 Å, α = {63.3}^{\circ}, t = 2017 Å$ . The estimated fit resolution was $4.3 Å$ and $4.2 Å$ without and with sample thickness estimation, respectively. (d) Thon ring fitting of a micrograph taken from plunge frozen rotavirus double-layered particles (Grant and Grigorieff, 2015). When fitting without thickness estimation the estimated parameters were $Δ f_{1} = 7027 Å, Δ f_{2} = 6808 Å, α = - {20.3}^{\circ}$ . When taking sample thickness into account the estimated parameters were $Δ f_{1} = 7027 Å, Δ f_{2} = 6808 Å, α = - {22.9}^{\circ}, t = 850 Å$ . The estimated fit resolution was $4.2 Å$ and $3.2 Å$ without and with sample thickness estimation, respectively.

Figure 4

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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 the RANSAC algorithm were manually inspected and color-coded according to visual inspection of the micrographs.

Figure 5

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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. The position on the x-axis corresponds to the thickness estimate by CTFFIND5. The black dashed line indicates identity. The red dashed line indicates the result of a linear fit.

Figure 6

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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 same micrograph as shown in panel (a) after CTF correction by phase flipping (c) or with a Wiener-like filter (d). The custom fall-off parameter was set to 1.3 and the custom strength parameter was set to 0.7.

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

Additional files

MDAR checklist: https://cdn.elifesciences.org/articles/97227/elife-97227-mdarchecklist1-v1.pdf
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Johannes Elferich
Lingli Kong
Ximena Zottig
Nikolaus Grigorieff

(2024)

CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples

eLife 13:RP97227.

https://doi.org/10.7554/eLife.97227.3

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