The Icebreaker software for ice gradient estimation and removal on cryoEM micrographs. External job for calling IceBreaker grouping within Relion 3.1 in_parts is input star file from relion picking job in_mics is previous grouping job directory Run in main Relion project directory PROCESSING THE MICROGRAPHS To process the micrographs use the ib_job script As an input, it takes a .star file with a list of micrographs to process, e.g. micrographs_ctf.star In the 'Params' tab, you have to add a label mode which can take values group (it will group areas with similar ice thickness) or flatten (it will improve contrasts locally trying to remove the ice gradient). In the Running tab use the slider to select the number of threads. The output from this job is a new set of micrographs. PROCESSING THE PARTICLES As 'Input micrographs' it takes micrographs 'grouped' with the previous script. As 'Input particles' any star file with particles from jobs like Extract particles, 2D classes, etc. It takes no additional parameters and this is single-threaded. As a result, additional parameter column (_rlnHelicalTubeID) with estimated ice thickness value for each particle will be added.