Extended Data Fig. 2: Cryo-EM reconstruction of ts2-active SERT–15B8 Fab–8B6 scFv–paroxetine complex.

a, Workflow of cryo-EM data processing of the ts2-inactive SERT–15B8 Fab–8B6 scFv complex with paroxetine in the outward-open conformation. After particle picking, particles were sorted using 2D classification. 3D ab initio reconstructions were performed after 2D classification using cryoSPARC. One out of two predominant classes (boxed) exhibited a subset of homogeneous particles that were used for further processing and global alignment in cryoSPARC. The other class, upon refinement, yielded only a nanometre-resolution map. Local refinement using cisTEM improved the resolution of class 1 (boxed) upon masking of the Fab constant domain and micelle (mask is shown overlaid in blue on top of the reconstruction). The final reconstructed volume was sharpened using PHENIX. b, Representative cryo-EM micrograph. Individual single particles are circled in white. Scale bar, 50 nm. c, 2D class averages after three rounds of classification. d, The angular distribution of particles used in the final reconstruction. e, Cryo-EM density map coloured by local resolution estimation. f, FSC curves for cross-validation, the final map (blue), masked SERT–Fab complex (red), and a mask that isolated SERT (black). The low-resolution limit cut-off for refinement was 7.5 Å. g, FSC curves for model versus half map 1 (working, red), half map 2 (free, black) and model versus final map (blue). h, Cryo-EM density segments of TM1 to TM12. i, A spherical mask placed over SERT was used for focused 3D classification with 3 classes. Comparison of the classes did not reveal any substantial differences. The antibodies were removed for clarity. The number of particles belonging to each class average is: class 1, purple (11.9%, 25,530 particles); class 2, yellow (54.9%, 117,781 particles); class 3, cyan (33.2%, 71,226 particles).