Extended Data Fig. 8: Cryo-EM processing schemes for Mot1 prehydrolysis state and Mot1^Δ50C:TBP:DNA dimer.

a) Processing of Mot1^wt:TBP:DNA with added ADP-BeF₃⁻ before SEC, representing the prehydrolysis state. Particles were picked with blob picker in cryoSPARC and sorted via multiple rounds of 2D classification. Particles from good classes were used for two times Topaz training with intermittent rounds of 2D classification. Dimer classes (marked by red asterix) were Topaz trained and sorted separately. A dimer ab initio model (C1) was calculated and non-uniformly refined to an average resolution of 4.4 Ȧ. Monomeric particles were submitted to 3D variability analysis followed by hetero-refinement of three distinct density maps. The map with the most distinct ATPase domain was non-uniformly refined to an average resolution of 3.6 Ȧ. To increase quality of Mot1^NTD and Mot1^CTD, both areas were masked separately and focused-refined to average resolutions of 3.7 Ȧ (C-terminal masked) and 3.5 Ȧ (N-terminal mask) respectively. b) Local resolution of the cryo-EM maps of Mot1 prehydrolysis complex from a). c) Fourier shell correlation of the masked cryo-EM map of Mot1 prehydrolysis complex from a). The ‘gold standard’ resolution cut-off (0.143) is marked by a dashed line. d) Processing of the Mot1^Δ50C:TBP:DNA dimer. For details on processing see Methods section. e) Local resolution of the cryo-EM maps of Mot1^Δ50C:TBP:DNA dimer from d). f) Fourier shell correlation of the masked cryo-EM map of Mot1^Δ50C:TBP:DNA from d). The ‘gold standard’ resolution cut-off (0.143) is marked by a dashed line.