Fig. 3: Cargo binding on the COPII inner coat.

a, Difference map between the STA of the inner coat on microsomes and a 14-Å low-pass-filtered volume representation of the fitted model of the inner coat from cargoless GUVs (PDB 8BSH) with Sar1 in yellow, Sec23 in blue and Sec24 in green. The Sec24 A and B cargo-binding sites are represented with pink blobs generated from bound cargo peptides described in previous X-ray crystallography studies (PDB 1PD0 and PDB 1PCX). b, As in a but using the STA map previously obtained from GUVs (EMD-15949) and low-pass-filtered to 14 Å. c, Schematic of the in vitro reconstitution of COPII budding from Sed5-enriched GUVs. Inset, details of 6xHis-tagged Sed5 associating to the Ni-NTA tagged lipids on the GUVs. d, XY slices through a representative tomogram of Sed5-enriched GUV budding reactions at different Z heights displaying the coated tube morphology (pink, Z = 162) and the inner (yellow, Z = 137) and outer (blue, Z = 128) coat arrangements. Scale bar, 100 nm. e, Detail of the STA map of Sed5-bound inner coat showing the region around the Sec24 B-site. Density closer than 3.5 Å to the fitted model of the inner coat (PDB 8BSH) is shown in green while white density corresponds to regions of the map that are not explained by the fitted model. The model of a Sec24-bound peptide from Bet1 cargo (PDB 1PCX), which contains the same B-site binding motif as Sed5 (LxxLE), is also fitted to highlight the location of the B-site and is shown in purple. White density in correspondence of this peptide is marked with an asterisk. f, As in e but displaying the map obtained from cargoless GUV reconstitution (EMD-15949).