# Single Particle Analysis: CTF determination

## 1. Automated CTF fitting and generating parameter files:

An automated CTF fitting can be run on all the micrographs, generating a power spectrum image file for each. The resultant fits need to be checked and refined in bshow:

bctf -v 7 -datatype float -action prepfit -sampling 2.2 -out klh_000239_fit.star klh_000239.star

## 2. CTF fitting in bshow

### 2.1 Setup

Depending on the option chosen in the previous section, start in the following way:

### 3.1.1. Existing parameter file for the micrograph:

Open the micrograph parameter file in bshow:

bshow klh_000239_fit.star &

Select the micrograph image to open. Then calculate the power spectrum (see below).

### 3.1.2. Using bshow directly:

Open the micrograph file in bshow:

bshow klh_000239.mrc &

Then calculate the power spectrum (see below).

### 3.1.3. Existing parameter file and calculated power spectrum:

The power spectrum is already calculated and an initial fit done. Open the micrograph parameter file in bshow:

bshow klh_000239.star &

Select the power spectrum to open.

### 3.2. Calculating the power spectrum

The micrograph image is opened in bshow, and the power spectrum is calculated (menu item “Image/Power spectrum”). The power spectrum can be calculated in different ways, but for fitting the CTF the best is to calculate tiles, and to average these to obtain a good estimate of the power spectrum (don’t calculate the logarithm of the power spectrum, as this would not allow an accurate baseline to be fitted). The default size for the tiles is typically sufficient, although smaller tiles will give a better estimate of the power spectrum with a decrease in the sampling of the radial power spectrum. The power spectrum can be saved for future reference (menu item "File/Save").

### 3.3. Fitting the CTF

Once the power spectrum is calculated, the CTF can be fitted in bshow (menu item “Micrograph/Fit CTF”). After setting the accelerating voltage, Cs (typically 2 mm ) and amplitude contribution (typically 0.2 for negative stain and 0.07 for vitrous ice) to the correct values, the quick fit button provides for a fast initial attempt to fit the radial power spectrum. If it does not give a reasonable initial fit, the user can manipulate the defocus until the first zero agrees with the first minimum in the radial power spectrum. The fit can be refined by successively going through the three buttons for baseline, envelope and defocus, each controlling only one aspect of the fit at a time. Once a good fit is obtained, the astigmatism button allows for an estimation of the astigmatism, only modifying the defocus deviation and astigmatism angle. The radial power spectrum is adjusted for astigmatism. At every stage, the user has full control over the fit and can improve it by hand. The extent to which the fitted CTF curve agrees with the radial power spectrum, gives the information limit. In other words, the point at which the last zero agrees with an apparent minimum in the radial power spectrum, gives the approximate maximum resolution with significant information above the background noise. This measure can be used to eliminate bad micrographs from further processing.