Usage
ctfit
In addition to determining the CTF parameters from experimental data, CTFIT can be used to simulate the effects of an electron microscope on simulated data. This can be used as a teaching tool to show what to expect as the microscope focus is changed, for generating simulated data for use in software testing, and to help predict what a specific molecule might look like on the microscope.
Area 1 is the list of currently loaded images and simulations. Each data set represents a power spectrum generated from one or more image files. When plotted, 2 lines will be displayed for each data set. One line is the power spectrum from the image. The other line is a simulation generated from the parameters specified in area 3 and 4. It is also possible to generate simulations without an associated data curve. When the program starts, one default simulation called 'New' will be automatically created and listed in this window. Each simulation and data set can be independently turned off and on (displayed in the plot window). Data sets currently displayed in the plot window will have a colored line to the left of them, indicating their color in the plot. To toggle the display of an image/simulation, simply double click on it in this list. Single clicking on an image/simulation will make it the current dataset. All of the parameters displayed in areas 3 and 4 refer to the currently selected data set in this list. If you select (single click) on another data set, all of the parameters in areas 3 and 4 will be updated. When a new data set is read into the program, the parameters will default to whatever their settings are when the image is loaded.
In areas 3 and 4 are all of the parameters for the currently selected data set/simulation. The first 11 items are sliders representing the parameters of the CTF/background model. The 5th parameter is experimental and is not used in CTF correction. The astigmatism parameters are also currently ignored by the CTF correction routine. It is suggested that you discard any images that exhibit any significant amount of astigmatism. Only 3 of the parameters in section 4 must be set for fitting results to be valid. Microscope voltage, Cs and A/pix (number of angstroms/pixel in the scanned image) must all be correct for the simulation/fit to be valid. The other parameters are stored for database purposes only (although it is suggested that you fill them in as you go to maintain a permanent record of your data).
The CTF correction model is described in detail in the CTF Correction section of the manual. To adjust the CTF parameters manually, simply drag the sliders or enter a number in the text box next to each slider. It is also possible to change the range of each slider. Simply enter '<' or '>' and the new minimum or maximum value for the slider.
Section 2 contains parameter memories. Each data set already has an independent set of parameters, but these values are not stored anywhere outside the program. If you quit, then restart the program, these parameters will all be lost. Parameter memories are used to provide a long term record of the parameters of any micrograph. They also provide a mechanism for tracking the performance of your microscope(s) over time. Memories are divided into groups for your convenience. Generally the groups represent different samples or conditions. To make a new group, type its name into the 'Group' selection box. The new group will the appear on the pull down menu. Any groups that are empty (no memories in them) when you exit the program will not appear when you next run the program. To create a new memory with the current parameters, select the Group you want the new memory to be in, then hit the 'New' button. A new memory will appear with the name 'Default'. Simply type the correct name in the box above the 'Set' button and press return to change the name. You can then use the the 'Set' and 'Rcl' buttons to change or restore the current parameters to/from the memory. The memories are persistent, ie - the next time you run ctfit, you will still have all of your memories from the last session. Double clicking on a memory is equivalent to selecting that memory and pressing 'Rcl'.
Sometimes, even more customization is required. Clicking on the plot with the middle mouse button (or both buttons at the same time for those without a middle button) will cause a plot inspector to appear. This window will allow you to customize the appearance of the plot. Note that many of the changes you make with the plot inspector may be transitory. ctfit has control of the plots, and will modify many of the settings itself if you do something that causes the plot to be redrawn. However, if you want to modify the plot appearance for printing or saving an image (both of these options are available in the plot inspector), you can make changes in the inspector then hit the 'print' or 'save' button immediately to save a snapshot of the plot. Note that the print option produces color postscript, which will produce much better output than saving a picture and printing that. Note that this plot inspector is still being developed, so some features are not fully functional yet (it will be obvious which).
The various plot types that can be displayed are:
CTF Parameter Determination
Actually determining the CTF parameters for a micrograph is not an absolutely trivial process. The difficulty lies in the fact that the measured data consists of the structure factor multiplied by the CTF. Generally speaking, the structure factor is unknown. Since it varies by several orders of magnitude, this can make accurate CTF parameter determination rather difficult. There are a few possible solutions to this problem :
Solution 1:
Collect x-ray solution scattering data for your specimen. Clearly this
is not a trivial thing to do. If you've done this, hit the 'From File'
button in the Structure Factor section of the screen. You can then read
your data into the program (it must be in a 2 column text file with scattering
intensity vs spatial frequency). Once you've done this, the simulation
line for the current data set will include the structure factor. You can
then try for an exact match between the simulation curve and your data
curve.
Solution 2:
The structure factor of proteins of similar size and general overall
shape are remarkably similar to each other on a logarithmic scale. While
there is some difference, perhaps a factor of 2 or 3 in the 5-10 A range
depending on helix/sheet content, the overall shape, which covers several
orders of magnitude, is basically the same. By using a 'standard' curve,
prepared from a set of similar proteins, the CTF parameters can be fit
fairly accurately in the same way as solution 1.
Solution 3:
This is the least reliable method, and generally using method 2 to
start out, then finishing off with method 3 is advisable. This method involves
realizing that the structure factor is always the same, regardless of defocus,
etc. This method is the reason for allowing 2 plots to be displayed simultaneously.
The idea is to get a good fit in the defocus and noise parameters in the
'Complete' plot, and simultaneously get all of the data sets to have the
same predicted structure factor in the 'Struc Fac' plot. This plot displays
a predicted structure factor for each data set, calculated by taking the
power spectrum minus the background divided by the CTF. Note that this
cuve will be very inaccurate when the CTF approaches zero, so it will
generally contain some large spikes near the zeros of the CTF. It will
also tend to diverge rapidly at high frequency due to inaccurate fitting
of the background. Neither of these effects represent a problem. Simply
ignore the spikes, and try to get the curves to match far from the zeros.
You might also try to get the same general behavior at high spatial frequencies.
The reason this method tends to be somewhat unreliable is that the envelope function width and the % amplitude contrast cannot be predicted accurately with this methodolgy. Generally a range of amplitude contrasts can be used, and a good match between data sets can still be obtained. This has profound effects on the final reconstruction. If you find you are getting a reconstruction with black or white 'bloctches' outside your map, chances are you have used the wrong amplitude contrast.
The envelope function is also a problem with this method. While this method does determine the relative envelope function between different data sets, it leaves the overall envelope function completely undetermined. To determine the overall envelope function, you have to have some knowledge of how rapidly the structure factor falls off, so the structure factor can be separated from the envelope function.
Both of these deficiencies can be taken care of using solution 2. Even if the match isn't perfect, using solution 2 to determine the amplitude contrast and envelope function width for one data set can 'bootstrap' using method 3.
Solution 4:
The last solution works only if you have collected some or all of your
data in ice on a continuous carbon substrate. Often this causes preferred
orientations, and the carbon film will effectively add more noise to the
data. However, with a known carbon film structure factor, you can box out
both areas of just carbon (no protein), as well as areas with protein.
These 2 types of data are then read in separately. The CTF parameters can
then be determined for the carbon film with a known carbon film structure
factor, then these same parameters can be applied to the protein (with
some possible small adjustments for defocus, since the protein is in a
different plane).
Performing CTF correction
Once the parameters have been determined, you need to actually apply
the corrections to your data. In EMAN, this occurs in 2 stages. The first
step occurs within CTFIT. Once you have determined the CTF and background
parameters for all of your data sets (and have stored them in individual
memories for later use), you then select each data set (1 at a time) and
then use the 'Phase Correct' menu item on each. This option will perform
the phase flipping portion of CTF correction, and will store all of the
CTF parameters in the image headers for use in the second part of CTF correction.
When you select this menu item it will read the original data set in, do
the phase flipping, then write the results to a new image file with '.fix'
inserted in the name. The '.fix' files are then used in reconstruction.
The second phase of CTF correction (CTF weighted amplitude correction)
is performed transparently by the reconstruction procedure. Simply enable
CTF correction with the 'ctfc=<res>' option.
Other options
There are a variety of other menu commands. These are currently undocumented.
Some are pretty self explanatory. Look for more details in future releases. One warning, if
you use the equation parser features, note that unary '-' has higher precedence than '^'. That is, if you want exp(-s^2), it must be entered as exp(-(s^2)).