Finding Defocus with Ctfplotter
for Three Example Tilt Series
(IMOD 4.9)
University of Colorado,
Boulder
This document will guide you through running Ctfplotter to find defocus for three
different tilt series, one where the signal of the CTF is very strong, one
where it is weak, and one where it is sometimes strong and sometimes weaker.
It will explain some of the most important aspects of
the process. For more details, consult the
Guide to Ctfplotter, which fully
explains each aspect of the interface and also has screen shots based on these
two data sets. Labels in the Etomo or 3dmod interface are shown in Bold, and entries in fields are shown in italics.
Determining Defocus in a Tilt Series from a K2 Camera:
This tilt series,
from Cindi Schwartz, is of a flagellum of a Giardia cell, taken with a K2 camera on
a Krios microscope at Janelia Farm. The total dose was 26 electrons/square
Angstrom. Images were taken in superresolution mode with an exposure time
of 0.5 sec to avoid having to save and align subframes, and reduced by a factor
of 4 with antialiasing. With this protocol, they may have somewhat better
high-frequency information than a typical tilt series taken in counting mode
without binning, so the power spectra may be particularly good here.
-
Download the sample data set from our web site.
-
Move the data set file "K2-CTF-Data.tar.bz2" to the directory where you want to
work on it. Its contents will unpack into a subdirectory named "K2-ctf".
-
cd to the directory with the file
- Enter the command:
imoduntar K2-CTF-Data.tar.bz2
or, anywhere except on Windows without Cygwin, you can use
tar -xjf K2-CTF-Data.tar.bz2
- Enter the data set directory with:
cd K2-ctf cd K2-ctf
- Start Etomo with the command
etomo WTI042413_1series4.edf
- Open the Final Aligned Stack page and switch to the Correct CTF tab.
Notice that a spherical aberration of 0 is allowed, and is
appropriate if data were collected on an aberration-corrected microscope,
as these were.
- The Expected defocus has already been set to 6.0 in this data set.
- The Config file has already been selected to access a configuration
file listing noise files in the "Janelia" subdirectory of the data set directory.
- Press Run Ctf Plotter. Note that the CTF is analyzed in the raw
stack, so the aligned stack does not need to exist before running Ctfplottter.
- The magenta curve shows the rotationally averaged power spectrum plotted versus
spatial frequency. The green curve is a fitted curve, which in general may
not fit well until fitting parameters have been adjusted.
- The units of spatial frequency along the X axis are reciprocal pixels and range
from 0 to 0.5/pixel.
- Power is always very high at low frequencies, so the first operation is to zoom
up the part of the curve that shows the CTF effect.
Click the mouse just under the hump in the curve after 0.1/pixel and drag the
zoom area out to the right edge, just under the curve, so that the Y axis range
is about -0.1 to 0.5.
- Since the fitting looks good already, switch to use the Current defocus estimate and
All tiles in the Angle Range and Tile Selection dialog and press
Apply.
- Press Fitting to open the dialog for setting fitting parameters.
- The
frequency of the first zero
determined from the fit is shown at the top of the plotter window after Z:,
and the corresponding defocus is shown.
It is ~4.6 microns instead of the nominal 6 microns. The fitting
range starts at a low frequency appropriate for the higher nominal defocus but too early
for this lower actual defocus. It is best if the fitting is done to a
linearly falling part of the curve and excludes the portion before that curving
away from a line. Change X1 Starts to 0.16.
- The fitting can go to the third zero or even the fourth, so change X2 Ends to
0.38 or 0.43. Press the Enter key or Apply to fit with a
changed value.
- Turn on Vary exponent of CTF function; the fit looks better, so that is
an appropriate parameter to include when fitting these data.
- To assess whether fitting to a single image is possible, go back to the
Angle Range and Tile Selection dialog, change the Starting
tilt angle
tilt angle to 0 and the Ending tilt angle to 1.8, and
press Apply. The curve looks good.
- To see whether this is still the case at high tilt, change the
Starting tilt angle to
60 and the Ending tilt angle
to 62 and press Apply. The curve still looks good.
- Turn on Fit each view separately and press Autofit All Single Views.
- Resize the Angle Range dialog so that you can see more of the table and
scroll through the values to see how much defocus varies. Note the changes
of over a micron up and down above 40 degrees. Double-click a series of
lines in the table to check the curve-fitting in this region. It is
clearly correct; the defocus changes from image to image have been measured
accurately.
- Press Save to File and exit Ctfplotter and Etomo.
Determining Defocus in a Tilt Series from a CCD camera:
This tilt series is of a preparation of bovine papilloma virus (BPV), taken
by Mary Morphew on
an F20 with a US4000 CCD camera at a nominal defocus of -3 microns. It is
the series used in the Ctfplotter Guide to illustrate program operations.
Here, it illustrates some of the challenges in doing CTF correction on
relatively low-defocus tilt series take with a CCD, which provides poorer
information at high frequencies than a direct detector.
-
If you already unpacked the data set for the SIRT tutorial, skip to "Enter the
data set directory".
-
Download the sample data set from our web site.
-
Move the data set file "CTF-SIRT-Data.tar.bz2" to the directory where you
want to work on it. Its contents will unpack into a subdirectory named
"ctf-sirt".
-
cd to the directory with the downloaded file
- Enter the command:
imoduntar CTF-SIRT-Data.tar.bz2
or, anywhere except on Windows without Cygwin, you can use
tar -xjf CTF-SIRT-Data.tar.bz2
- Enter the data set directory with:
cd ctf-sirt
- Start Etomo with the command
etomo bpv_-3_3a.edf
- Open the Final Aligned Stack page and switch to the Correct CTF tab.
- As for the K2 data set, the Config file has already been selected to
access a configuration file listing noise files in the "F20" subdirectory of the
data set directory.
- Set the Expected defocus to 3.0.
- Press Run Ctf Plotter.
- If you press the help icon in the upper right of the Ctfplotter window, it will
bring up the Ctfplotter Guide; the Examples section there shows screen shots
from this data set.
- The power spectrum has two dips in the falling part of the curve, neither of
which correspond to zeros. The dip at the first zero is not evident until
the curve is zoomed. Zoom it up by selecting the top of the second hump in
the magenta curve and dragging the selection region to a point before a frequency of
0.4/pixel and just below the baseline.
- Select All tiles and press Apply. This reduces the noisiness
of the curve.
- Double-click the left mouse button at the first zero (frequency 0.25/pixel).
The defocus readout on the top line changes to show the defocus corresponding to
this point, 3.6 microns.
- Press Fitting to open the Fitting Range dialog. The fitting range
set from the nominal defocus is particularly inappropriate in this case.
Set X1 Starts to 0.185 to fit the falling phase of the power
spectrum back to where its slope changes. Set X2 Ends to 0.29 because the signal falls off there, well before the location of the
second zero. Press the Enter key or Apply to fit with the changes.
- You can now zoom up another step to make it easier to see the CTF signal.
Select a point just above where the two curves diverge on the left and drag the
selection box out to the right edge under the baseline.
- Select Current defocus estimate in the Angle Range dialog now that the
fitting is being done to an appropriate range.
- Now that fitting is set up, you can press Autofit All Steps to fit to 40-degree ranges at 20 degree intervals.
- Double-click each line in the table to check the result. The dip in the
fitted curve should seem like a reasonable fit to the data, except possibly for
the last angular range. If you think that the zero is not correctly found
by the fit, double-click where you think the bottom of the dip is located, and
press Store Defocus in Table to replace the value from the fit.
- Press Save to File and exit Ctfplotter and Etomo.
- If you are going to do the practice SIRT reconstruction with this dataset,
or if you want a corrected stack for any other reason, then select up to 12 CPUs
in the parallel processing table, and press Correct CTF then Use CTF Correction when it is
done.
Determining Defocus in a Tilt Series from a DE-12 Camera:
This tilt series,
from Cindi Schwartz, is of a preparation of microtubules decorated
with the motor protein Eg5,
taken with a DE-12 camera during a demo on the F20 microscope in Boulder. The
tilt series had a 2 degree increment and the total dose was
79 electrons/square
Angstrom. Parts of the series have good signal for determining CTF, but
not all of it.
-
Download the sample data set from our web site.
-
Move the data set file "DE-CTF-Data.tar.bz2" to the directory where you want to
work on it. Its contents will unpack into a subdirectory named "DE-ctf".
-
cd to the directory with the file
- Enter the command:
imoduntar DE-CTF-Data.tar.bz2
or, anywhere except on Windows without Cygwin, you can use
tar -xjf DE-CTF-Data.tar.bz2
- Enter the data set directory with:
cd K2-ctf cd K2-ctf
- Start Etomo with the command
etomo MTEg5series5D.edf
- Open the Final Aligned Stack page and switch to the Correct CTF tab.
- The Expected defocus has already been set to 6.0 in this data set.
- The Config file has already been selected to access a configuration
file listing noise files in the "DE12-Div2" subdirectory of the data set directory.
- Press Run Ctf Plotter.
- Zoom the power spectrum by clicking on the magenta curve to the left of
0.1/pixel and dragging the selection region to a point before a frequency of
0.4/pixel and just below the baseline.
- The power spectrum shows a clear signal out to the third zero and the green
fitted curve matches the location of the zeros fairly well, so the defocus
estimate is good. Select both All Tiles and Current defocus estimate and press
Apply.
- Press Fitting to open the Fitting Range dialog. Set X1 Starts
to 0.1, since the fitted curve deviates before this point. Set
X2 Ends
to 0.25 to fit out to the third zero, and press the Enter key or Apply.
- Turn on Vary exponent of CTF function; the fit does not look any better
so there is no reason to leave this option on. Turn it off for now; below
you will see how it is inappropriate in some cases.
- To see if single images can be fit, set the Ending tilt angle to -18
and press Enter or Apply. This curve is noisy but the
fitting still seems reasonable.
- To see if fitting is still good at high tilt, change the
Starting tilt angle to
-60 and the Ending tilt angle
to -58. The curve is even noisier but fitting is still plausible.
- Turn on Fit each view separately and press Autofit All Single Views.
- Resize the Angle Range dialog so that you can see more of the table and
scroll through the values. Notice that there are some big jumps at
positive tilt angles, particularly from 25 to 27 and from 41 to 43.
Double-click these lines in the table to see these fits. The power spectra
at 27 and 43 are particularly low in signal, so fitting to the noisy data from
single views is just not reliable.
- To get a better sense for how often the fitting looks good, double-click other
lines through the series. At most (but not all) negative tilt angles, the
fit looks fairly reliable; but at positive tilt angles it often is not.
Since inaccurate defocus values can do more harm than good, we need to fit to
multiple views instead, with the reduced goal of estimating the trend in defocus
through the series.
- Now switch to fitting sets of 4 views (8 degree ranges) by entering a
Starting tilt angle of 0, an Ending tilt angle of 8,
and 4 for Step angle range by. Turn off Fit each view
separately and press Autofit All Steps. The program will ask
you to confirm that you want to replace all of the existing values in the table;
press Yes.
- Double-click the lines at positive tilt angles to see how reliable these fits
look. The signal gets rather low at the highest tilts, but the noise is
low enough to allow you to see that the defocus is being found adequately.
- To see the potential problems with adding the exponent of the CTF function as
a variable in the fit, double-click the line for 21 to 29 degrees. Press
Apply 5-10 times and watch the defocus value (D:) in the plotter window.
It varies from 5.26 to 5.29, which means that fitting is self-consistent and not
too sensitive to the current assumed defocus value. Now turn on Vary
exponent of CTF function in the Fitting Range dialog and
press Apply many times in the Angle Range dialog. The estimated
defocus jumps around between 5.15 and 5.3, a 5-fold bigger range. When
the signal is too weak, adding this fifth parameter to the fit can significantly
reduce the reliability and stability of the fits.