Extended Data Fig. 2: Cryo-EM processing pipeline.

Flow chart showing the image-processing pipeline for the cryo-EM data of Cdiff Eσ^A/Fdx complexes, starting with 6,930 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K3 Summit direct electron detector (Gatan). Movies were frame-aligned and summed using MotionCor2⁴¹. CTF estimation for each micrograph was calculated with cryoSPARC2⁴². A representative micrograph is shown following processing by MotionCor2⁴¹. Particles were auto-picked from each micrograph with cryoSPARC2⁴² Blob Picker and then sorted by 2D classification using cryoSPARC2 to assess quality. The selected classes from the 2D classification are shown. After picking and cleaning by 2D classification, the dataset contained 2,415,902 particles. A subset of particles was used to generate an ab initio templates in cryoSPARC2 and 3D heterogeneous refinement was performed with these templates using cryoSPARC2⁴². One major, high-resolution class emerged, which was polished using RELION⁴³ and further cleaned with two more 3D heterogenous refinements. The final 182,390 particles were refined using cryoSPARC Non-Uniform refinement⁴⁴.