Icebreaker: Software for High Resolution Single Particle CryoEM with Nonuniform Ice

With many software tools available the optimal particle selection is still a vital issue in the single particle cryoEM. Regardless of the methods used, most pickers struggle when the varying ice thickness is present on the micrograph. We present IceBreaker, which allows us to estimate the relative ice gradient and flatten it based on the K-Means clustering algorithm, thus equalizing the local contrast. It allows differentiation of the particles from the background, and improves the particle pickers performance. Furthermore, a new parameter corresponding to the local ice thickness is introduced for each picked particle. Particles with a defined ice thickness can be grouped, sorted, and filtered based on this parameter during processing. Single particle 3D reconstructions can be made from particles in each ice group to access the effect of ice thickness. These functionalities are especially valuable for on-the-fly processing to automatically pick as many particles as possible from each micrograph and to select optimal ice regions for data collection. The software can be also used to evaluate the quality of the collected data, and also of already refined maps, deposited with the coordinates of the selected particles and to assess how the particles from different ice thickness areas contributed to the final map. Finally, the estimated ice gradient distributions can be stored separately and used to inspect the general quality of prepared samples.