We first averaged the density of the three heterotetramers with each rhombic region (see Fig. 1c). We used Coot 34 and REMO 35 to build the atomic models for E and M proteins based on this averaged density map. The protein backbone was first traced with the ‘baton’ tool in Coot. The resulting Cα model was converted into a full-atom model with REMO. We used the CNS package 36 (link) to refine the E:M:M:E heterotetramer structure by pseudo-crystallographic methods as previously described 37 (link) with its two-fold symmetry as an non-crystallographic symmetry (NCS) restraint. One half of the tetramer, containing one copy of E and one of M, was fitted into the density of each of the three copies of E and M in an asymmetric unit. The resulting atomic model was further refined by the CNS package 36 (link) against the map of the entire virion, with icosahedral symmetry as an NCS constraint.
We then built atomic models for the glycans at Asn67 and Asn153. Atomic models for a single sugar of N-acetyl-glucosamine (NAG) and a disaccharide with two NAGs were built for Asn67 and Asn153, respectively. Densities for additional sugars on these two glycosylation sites exist but are poorly ordered and were therefore not modeled. These additional sugars are more apparent in lower resolution density maps, suggesting their flexibility.
The full model was refined again in CNS as described above (R-factor: 29.3%, see R-factors of individual resolution bins in Supplementary Table 2). We also added sugars to the atomic model for the averaged tetramer and refined that model. The final R-factor for the averaged tetramer is 28.8% at 3.5 Å.