(A) Cryo-electron microscopy reconstructions of COPII cages assembled in vitro in the presence of inner coat subunits, but in the absence of a membrane, showed additional density below that of the outer coat. The top panel shows the density for the inner coat obtained in cages formed in the presence of Sec23 alone (Bhattacharya et al., 2012). The bottom panel shows the density for the inner layer in cages formed in the presence of Sec23/24 (Stagg et al., 2008). The position of the outer coat is marked as a blue outline. Note that in both studies, the twofold symmetry of the outer coat vertex was imposed on the inner coat. (B) Based on the comparison between the structures in panel A, Stagg et al. proposed that Sec23 binds at two positions: one copy below each vertex and two copies at its side. Sec24 would be located under the holes in the cage (Bhattacharya et al., 2012). According to this model, the inner coat arrangement does not conform to the twofold symmetry applied during the reconstruction procedure, explaining why it is poorly resolved in the cage reconstruction. (C) A previous model was proposed based on the reconstruction of icosidodecahedral cages (bottom panel in A). In this model, Sec23/24 subunits are oriented with their long axes in the direction of the +/+ connection of the outer coat vertex. Two arrangements consistent with this model are shown: in the first (top) the subunits all have the same orientation, in the second (bottom), subunits have opposite orientations, following the twofold symmetry of the outer coat. (D) A schematic representation of the relationship between inner and outer coat as identifed in this study. This arrangement is similar to the model suggested in C (top). The stoichiometry of the two coat layers is 1:2 outer:inner in coated tubes, meaning that only half of the inner coat subunits are bound by the known Sec31 interface. The percentage of bound inner coat subunits would be higher on spherical vesicles. The difficulty in resolving the position of the inner coat in the studies of cages assembled in the absence of a membrane can be explained by our observation that the relative positions of outer and inner coats are constrained but not fixed, and that the two layers do not follow the same symmetry.