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ProtCTFFind

Jose Miguel de la Rosa Trevin edited this page May 21, 2016 · 4 revisions

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grigoriefflab - ctffind

With this protocol you can calculate the PSD (Power Spectral Density) and estimate the CTF parameters (defocus U, defocus V, defocus angle, etc) for a set of micrographs using ctffind ([1],[2]). The algorithm consists of computing a PSD from each input micrograph, estimating the PDS’s background, subtracting it from the original PSD, and evaluating the similarity between theoretical CTF functions and the remaining oscillatory signal.

Input parameters

To estimate the CTF you will need to select first the Input micrographs, that can be any SetOfMicrographs produced earlier (Fig. 1). Then you can choose the frequency region to be analyzed. The limiting frequencies must be such that all zeros of the PSD are contained within those frequencies. There is a wizard, shown in Fig. 2, that helps in choosing those frequencies. To see the full available options, choose the Advanced expert level and click on the Help button for any specific parameter (Fig. 3). The CTFFind protocol allows to use either the ctffind3 or ctffind4 program (the latest has been reported to be about ten times faster than its predecessor).

01.CTFFind
Figure 1. GUI input form of the ctffind protocol


02.CTFWizard
Figure 2. CTF wizard helps to select a downsampling factor and the range of spatial frequencies


03.CTFFindAdvanced
Figure 3. Advanced protocol parameters

Analyzing CTF results

The CTFs of good micrographs typically have multiple concentric rings, shown in Fig. 4 left, extending from the image center towards its edges. Bad micrographs may lack rings or have very few rings that hardly extend from the image center. A reason to discard micrographs may be the presence of strongly asymmetric rings (astigmatism, Fig. 4 center) or rings that fade in a particular direction (drift, Fig. 4 right).

04.CTFexamples
Figure 4. CTFs of good, astigmatic and drift micrographs respectively.


When the protocol is finished you may click on the Analyze Results button to show the list of micrographs and their PSD (Fig. 5). To discard micrographs with bad CTFs you may click with the mouse right button and press Disable. Once you finish the selection, press on the Micrographs button to create a subset of micrographs with only the enabled ones.

05.CTFResult
Figure 5. Displaying CTF results


Sometimes the CTF estimation algorithm may fail to find the rings even if they can be seen by eye. If this is the case, you may help the algorithm to find the rings by clicking on the image with the mouse right-button and choosing Recalculate CTF on the menu that appears. A graphical interface will help you to correctly identify the CTF. You must provide the first CTF zero and the search range, and then press OK. When you finish, press the Recalculate CTFs button.

It is possible to analyze the CTF profiles by right-click on a micrograph row and selecting the Show CTF profile option which should open a window shown in fig. 6. It is also possible to analyze the CTF fitting by selecting the Display CTF fitting option. A plot should appear with the 1D profiles calculated by ctffind4 (fig. 7). This is an interactive plot that can be zoomed to specific regions among other things.

06.CTFProfile
Figure 6. CTF profile


07.CTFFitting
Figure 7. CTF fitting


References

  • [[[1]]] Mindell, J. A. and Grigorieff, N. (2003). Accurate determination of local defocus and specimen tilt in electron microscopy. JSB, 142: 334 – 347.

  • [[[2]]] Rohou, A. and Grigorieff, N. (2015). CTFFIND4: Fast and accurate defocus estimation from electron micrographs. JSB, 192: 216 – 221.

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