Preparing the power spectrum
To fit the CTF, first read in the micrograph image using the "File/Open" menu item, or read a STAR parameter file using the Micrograph/Read parameters menu item. Make sure the pixel size (middle right side) is set correctly.
Then calculate the power spectrum using the "Image/Power spectrum" menu item. If a micrograph is open choose the "Average multiple power spectra" and "Tiles" options, and set the tile size to the desired size. If a stack of particle images is open, only choose the "Average multiple power spectra" option.
If the micrograph is tilted, set the tilt axis angle and the tilt angle. The tilt axis angle is taken as the counter-clockwise rotation angle starting from the x-axis. If the tilt angle is non-zero, tiles only along the tilt axis will be extacted.
Determining the CTF parameters
Select the "Micrograph/Fit CTF" menu item - this will open a new window used for CTF fitting.
Layout of the CTF fitting window
green: baseline
orange: envelope
red: calculated RPS (including CTF)
blue: radial power spectrum (RPS)
The RPS is calculated from the power spectrum with consideration of the astigmatism.
Plot controls
The origin of the plot can be set in screen pixel units relative to the lower-left corner. The scale can be set to expand the plot horizontally and vertically. The resolution limits (vertical green lines in the plot) can be set and are used to calculate measures of fit.Fitting the CTF
All of the curves except the radial power spectrum (blue) are updated continuously as the scale bars are changed. The radial power spectrum is updated on clicking the "updateCTF" button in the bottom left corner. In some entry boxes, pressing the return key will also change the radial power spectrum.The defocus deviation is not allowed to be larger than 90% of the defocus average.
The baseline and envelope equations can be changed to any
form. The
independent variable is the reciprocal space distance, s, which must be
written as "$s" or its square as "$s2". Valid equations are for
example:
0.2*exp(-5000*$s2) +
0.14*exp(-300*$s2)
0.2*exp(-1000*$s2)
The fitting typically proceeds with the "Fit" buttons from the left to the right. The "Quick" button does a quick fit without astigmatism and should always be done first and only once. The "Baseline", "Envelope" and "Defocus" buttons refines the fit for each separately and can be used multiple times in any order. The "Astigmatism" button starts an attempt to refine all parameters at the same time - this is a very unstable fitting approach and the result should not be blindly accepted!
It is important to finally refine the fit by hand and make
sure the parameters are reasonable.
The R value should not be taken as a definitive measure of fit. A better measure of fit is to find those CTF parameters that best fit the minima or zeroes of the CTF curve.
Reading and saving CTF parameters
The "Micrograph/Read parameters" and "Micrograph/Write parameters" menu items are used to read and write parameter files (usually in the STAR format).