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Hi all, I'm concerned about whether a particle set is being normalized properly during particle extraction in Relion 1.4. Attached is a star file of representative particles (icosahedral virus). At the bottom is my particle extraction command. Backgrounds seem to vary quite a bit, even when boxes are not over the edge of the micrograph. I should say a good number of the micrographs show a regular light/dark gradient across the image (due to acquisition issues), and that each contain at least a single row of "dead" pixels. Particles were extracted from raw micrographs following CTF estimation in Relion using .box coord files determined in EMAN2, so these images should not have any normalization done to them except that specified by Relion's --norm option. Concerned that intensity variation will be problematic downstream -- can anyone suggest Relion-compatible methods to take into account the background gradients (if that's the culprit), or am I doing something more obviously wrong? Thanks for any input! Command for particle extraction: -bash-4.1$ srun --nodes 5 --ntasks-per-node 24 relion_preprocess --o ptcls --mic_star micrographs_ctf.star --coord_suffix ".box" --extract --extract_size 384 --norm --bg_radius 190 --white_dust -1 --black_dust -1 --coord_files "Micrographs/*.box" -- particles themselves are approximately 260A diam -- also attached an example of early 2D classes showing background variation Thanks!!