Determining the CTF
For a general introduction to the contrast transfer function (CTF) see this link.
A prerequisite is that the tilt axis and tilt angles are known to a reasonable accuracy. The nominal axis and tilt angles should be sufficient to get good power spectra for CTF fitting. The more accurate angles determined from alignment is then used during reconstruction.
As with micrographs processed for SPA, the image is tiled, each tile transformed, and the resulting power spectra averaged to give a relatively noise-suppressed spectrum. The complication is to adjust for the specimen tilt that causes a defocus gradient across the image. The tilt angles and tilt axis must therefore be specified in the parameter file to calculate the correct defocus for each tile. The best workflow is to align the tilt series first to get accurate tilt and axis angles. In addition, an approximate defocus for the whole tilt series need to be given. The CTF parameters are then determined for each micrograph in the tilt series. If the nominal defocus provided in the beginning is too far off the determined values, the whole process can be repeated with the defocus values in the parameter file.
The following command line will set up the initial CTF parameters, prepare the average power spectra, and determine CTF parameters:
bctf -verb 1 -act prepfit -tile 256,256,1 -Volt 300 -Defocus 2.6 -Amp 0.07 -resol 10,50 -out tomo_ctfit.star tomo.star
The tile size should be chosen to give reasonable Thon rings in the power spectra. The tile edge size should be:
As always with CTF fitting, the quality of the fits should be checked in bshow.
Correcting for the CTF
The CTF of the micrographs can be corrected before or during reconstruction. The same types of CTF correction are available in both cases.
The CTF can be corrected in several ways, from the conservative flipping phase to the most aggressive correction algorithms. A good choice (at least to start with) is to flip the phases where the CTF curve is negative:
bctf -verb 1 -act flip -tile 128,128,1 -mic -out tomo_flip.star tomo_ctfit2.star
The tile size for CTF correction should be relatively small, but not too small to avoid poor spectral sampling. It may be necessary to find a good size for a particular case or a particular tomographic setup.