3D variability analysis and non-uniform refinement in Cryosparc

For the data refinement and analysis in Cryosparc, the Relion particle stacks from the global consensus refinement as well as the consensus refinement map (as reference volume) were imported into the cryosparc v2 pipeline⁶³. A consensus refinement in Cryosparc using the Homogeneous refinement tool was produced, which was used as an input for the 3D variability analysis⁶⁴. For the variability analysis, the wide mask created automatically during refinement in Cryosparc (including micelle) was applied. The frames of the 3 principal components generated in the 3D variability analysis were visualized using the ChimeraX volume series tool and shown in the recorded movies. Backbone models of the Cryosparc 3D variability frames were modelled into the extreme frames (#0 and #19) of each component using Isolde, based on the final consensus model. The Cryosparc non-uniform refinement tool⁶⁵ was used as alternative refinement method, to test resolvability of lower resolution areas, using default parameters and particle stacks imported from the Relion consensus refinement. The output maps were used to validate the Relion-derived maps and models and deposited as additional maps.