Preprocess images

RELION will work best if your data are

• 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 rectangle/B-spline).
  • Xmipp implements the Fourier-space downscaling in the xmipp_scale program with the -fourier option.
• 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, that's a very bad thing to do in general: go back to the original data.
• Normalised (the exact procedure does not matter too much, as errors in the normalisation are corrected internally)
• In one or more MRC stacks (with extension .mrcs; but also SPIDER and IMAGIC files may be read)

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 (take great care in sample preparation and data collection)