Preprocess images

RELION will work best if your data are

• Even-sized (Odd dimensions are not implemented)
• Clean from false particles (no images are discarded during refinement).
  • Xmipp implements an image sorting utility called xmipp_sort_by_statistics that is very handy in the cleaning of a data set.
• Unmasked (masking is performed internally)
• Non-interpolated (prevent any prior rotations/translations: use the originally scanned pixel values)
  • If downscaling is necessary because of memory issues: use a window-operation in Fourier-space, not a convolution in real-space (e.g. with a rectangle/B-spline).
• Uncorrected for CTF (this is done internally)
  • If your data have previously been phase-flipped, that's OK: just tell RELION about it
  • Actually, if you are not planning to correct for CTFs inside RELION (e.g. for negative stain data), phase-flipping is recommended.
  • If your data have previously been pre-Wiener filtered or pre-multiplied by their CTF, that's a bad thing to do: go back to the original data.
• Normalised Make sure the average density in the background area is (approximately) zero!!. Also, the standard deviation in the noise should be (approximately) one.

And then, just like with any other refinement program, you might save yourself lots of trouble if your data have:

• high signal-to-noise ratios (get the best possible structure by taking great care in sample preparation and data collection)

Recommended preprocessing procedure

1. Get a MRC/SPIDER/IMAGIC stack of your clean, unfiltered, unmasked and non-CTF-corrected images (phase-flipping alone is OK). Often direct extraction from the original micrographs is the easiest and best option.
2. If you want to correct for the CTF inside RELION (highly recommended for cryo-EM data, often less necessary in case of negative stain data) then create an input STAR file with the necessary CTF parameters for each particle
3. Use the relion_preprocess command-line program (available from version 1.1 onwards) to re-scale and re-window your original stack to even-sized particles (if necessary) and to normalize them. The output MRC stack and STAR file from this program may directly be used for 2D or 3D refinement in RELION.