An x-ray crystallographic-derived atomic model (PDBID: 2CIH) was fitted to the eight maps obtained with different data acquisition conditions using the “fit in map” function in UCSF Chimera (1.11.2)48. The maps were segmented using SEGGER (v1.4.9)49 and an α-helix corresponding to residues 48–77 was visualized for each map. FSC curves of each of the data sets were calculated. The correlation between half-maps (GS-FSC)34 was estimated by 0.143 cut-off50, and the correlation between map and atomic model (PDB ID: 2CIH) (MM-FSC) was calculated with 0.5 cut-off. The PDB-based atomic model map was generated using UCSF Chimera's “molmap” function48 at the GS-FSC resolution of each reconstructed map. The CTFFIND estimated resolution was assessed by analysis of the logfiles generated by CTFFIND46. B-factor plots were calculated by running an appropriately modified copy of RELION's script, bfactor_plot.py. The script randomly selects subsets of particles from each data set and executes Refine3D and Postprocess steps. Plotting the natural logarithm of each particle subset against the inverse of the squared resolution for each refinement allows estimation of particle and dataset quality by linear fit correlation11.

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