From Micrographs to Particle Stack ("Preprocessing")

Assumptions:

  1. The micrographs are MRC files, called "img_0001.mrc", "img_0002.mrc", ..."img_0010.mrc".
  2. The tight radius of the particles is ~30 pixel, after 2 X 2 binning of the micrographs.
  3. A box with the dimension of 80 pixel can comfortably accommodate the particles after 2 X 2 binning.

Procedure:

  1. Make a folder called "mrc-files", and put all micrographs in there.
  2. Make a folder called "dat-files_bin2", go into it, and run "sampilcopy2d.py ../mrc-files/img_####.mrc img_####.dat 1-10 0,0,A2,0".
    The resulting 2 X 2 binned micrographs are named as "img_0001_bin2.dat", etc. (Binning is done by averaging the neighboring pixels.)
  3. Pick particles from the binned microgrphs, and the particle coordinate files are called "SVCO_img_0001_bin2.dat", etc.
    Make a folder called "svco-files_bin2", and put all coordinate files in there.
  4. Make a folder called "cutprep", go into it, and run "sampilboxparticle.py ../dat-files_bin2/img_####_bin2.dat ../svco-files_bin2/SVCO_img_####_bin2.dat 80 1-10" when prompted, input the following parameters:

       >>>> Output particle stack name (no extension) : stack
        >>>> Output particle stack format SPIDER/MRC (S/M) : S
        >>>> SIGMA range to remove outliner (e.g. 5; 0: skip) : 5
        >>>> Invert contrast? [y/n or 0] : 0         >>>> Bin particles using Fourier interpolation to size (in pixel; 0: skip) : 0
        >>>> Normalization multiplier (Integer. e.g. 1; 0: skip normalization) : 1

The resulting stack is called "stack_fixs5_n1.dat".

Notes:

(1) For high resolution cryo-EM work, one should always box out particles from the original micrographs without binning.
In the step(4) above, run "sampilboxparticle.py ../mrc-files/img_####.mrc ../svco-files_bin2/SVCO_img_####_bin2.dat--2 160 1-10"
when prompted, input the same parameters as above. The resulting stack "stack_fixs5_n1.dat" is "original" particle stack (160 box size).

This stack can, for example, be 2 X 2 binned by running "sampilcopy2d.py stack_fixs5_n1.dat stack_fixs5_n1.dat all 0,0,F80,1".
Here, the binning is done using Fourier cropping, which is slower but preserves the most information without introducing artifacts.
The resulting stack is called "stack_fixs5_n1_fp80_n1.dat".