Supplementary Figure 1: Cryo-EM reconstruction of ASCT2.

a,b, Representative cryo-EM image (a) and 2D-class averages (b) of vitrified ASCT2. c, Angular distribution plot of particles included in the final C3-symmetrized 3D reconstruction. The number of particles with the respective orientations is represented by length and colour of the cylinders (long and red: high number of particles; short and blue: low number of particles). d, Image processing work flow. e, Analysis of conformational heterogeneity in the dataset. 3D classification without any symmetry imposed where performed on the indicated particle dataset: after several rounds of 2D classification (I, on 628,015 particles) and after several rounds of 3D classification (II, on 307,619 particles). Particles obtained before particle polishing, which rendered a map of 3.9Å resolution were used to perform symmetry expansion followed by particle subtraction on the individual protomers, yielding a dataset of 552,240 particles (III). Resulting classes of the 3D classification without image alignment are shown. In all cases only the inward-facing state of ASCT2 was identified. f, Final reconstruction map coloured by local resolution as estimated by Relion. g, FSC plot of the final refined unmasked (grey) and masked (blue) map. The resolution at which the curve drops below the 0.143 threshold is indicated. A thumbnail of the mask used for FSC calculation overlaid on the atomic model is shown in the upper right corner. h, FSC curves of the refined model versus the map of ASCT2 for cross-validation. The purple shows the FSC curves for the refined model compared to the full masked dataset (FSCsum); light grey, FSC curve for the refined model compared to the masked half-map 1 (FSCwork, used during validation refinement); dark grey, refined model compared to the masked half- map 2 (FSCfree, not used during validation refinement). Dashed lines, FSC threshold used for FSCsum of 0.5 and for FSCfree/work of 0.143.