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
  • 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 a MRC stack (although 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)