Extended Data Fig. 5: Cryo-EM data processing of the α3-α4-CsRubisco map (PDB: 8Q05).

a, Sharpened (B-factor: 45) post-processed map of the C1 symmetry-refined α3-α4-CsRubisco complex using the D4 symmetry expanded particle dataset. The soft featureless mask used for the classification is shown in purple, over the region of additional density, into which the predicted helix of α3 is built. Shown at a contour level of 0.0496. The 3D class (class 3) from which the particle dataset was D4 symmetry expanded is shown inset, and the mask is schematically represented over a region of additional density. b, C1 symmetry 3D classification of sub-particles using the soft featureless mask. The selected class with α3-α4 density is shown in blue, with the discarded classes in grey below. At this resolution, the density shows clearly helical nature. c, Second round of 3D classification using the selected sub-particles from the first round. The selected sub-particles from this round were used for reconstruction of the α3-α4-CsRubisco map. d, Phenix local resolution estimate of the α3-α4-CsRubisco map, shown at a contour level of 0.0293. e, Map density of the α3-α4 region in the unsharpened, post-processed C1, symmetry expanded map shown at contour level 0.0293 (top), and the unsharpened, post-processed D4, non-symmetry expanded map, shown at a contour level of 0.0174. Both maps are carved with a radius of 2 around the modelled helical region. f, Histogram showing the distribution of α3-α4 occupancy in the sub-particles of the particles used for the reconstruction of the α3-α4-CsRubisco map. g, Fourier shell correlation (FSC) curve showing the resolution estimate for the α3-α4-CsRubisco map with FSC cut-off of 0.143 (dashed lines). h, Model of α3-α4 in the density displayed with the side chains of residues with no density support displayed in green (top) and removed in the final coordinates (bottom). i, Coordinates at the α3-α4-CsRubisco interface displayed in the map density at a contour level of 0.0304 and carved with a radius of 2. j, Nomenclature of RbcL regions at the α3-α4-CsRubisco interface. k, A potential hydrogen bond network between Gln170 of α3-α4 and Glu93 and Gln95 of the CsRbcL CD loop shown with and without map density shown at a contour level of 0.0396. l, α3-α4-CsRubisco interaction map.