3.1.  The ensemble map and the way to obtain it  
In the following, we describe the analysis of SARS-CoV-2 spike stabilized in the prefusion state by two proline substitutions in S2 (S-2P). We will objectively demonstrate that the flexibility of the spike protein should be understood as a quasi-continuum of conformations, so that when performing a structural analysis on this specimen special care has to be paid to the image-processing workflows, since they may directly impact on the interpretability of the results.
Starting from the original SARS-CoV-2 S-2P data set of Wrapp et al. (2020 ▸), we have completely reanalyzed the data using our public domain software integration platform Scipion (de la Rosa-Trevín et al., 2016 ▸), breaking the global 3 Å resolution barrier. A representative view of the new ensemble map and its corresponding global FSC curve is shown in Fig. 1 ▸(a) (EMDB entry EMD-11328); the sequence of a monomer of the S protein is shown on the right to facilitate the further discussion of structure–function relationships (from Wrapp et al., 2020 ▸). Figs. 1 ▸(b) and 1 ▸(c) show a comparison between the original map (Wrapp et al., 2020 ▸) with EMDB code EMD-21375 and the newly reconstructed ensemble map corresponding to EMD-11328. Clearly, the local resolution (Vilas et al., 2018 ▸), which is shown on the left in Figs. 1 ▸(b) and 1 ▸(c), is increased in the new map, and the anisotropy, which is shown in the center, is much reduced. Finally, on the right we present plots of the radially averaged tangential resolution, which is related to the quality of the angular alignment (Vilas et al., 2020 ▸); the steeper the slope, the higher the angular assignment error. As can be appreciated, the slope calculated from the newly obtained map is almost zero when compared with that for the map from Wrapp et al. (2020 ▸), indicating that, in relative terms, the particle alignment used to create the new map is better than that used to build the original map. The result is an overall quantitative enhancement in map quality.
In terms of tracing, besides modeling several additional residue side chains and improving the geometry of the carbon skeleton (see Supplementary Fig. S2), one of the most noticeable improvements that we observed in the new map is an extension of the glycan chains that were initially built, particularly throughout the S2 fusion subunit (PDB entry 6zow). A quantitative comparison can be made between the length of glycan chains in the new ‘ensemble structure’ with respect to the previous structure (PDB entry 6vsb; see Supplementary Table S2). Although the total number of N-linked glycosylation sequons throughout the SARS-CoV-2 S trimer is essentially the same in the new structure (45) and PDB entry 6vsb (44), we have substantially increased the length of the glycan chains, expanding the total number of glycans by about 50%. We note the importance of this extensive glycosylation for epitope accessibility and how the accurate determination of this glycan shield will facilitate efforts to rapidly develop effective vaccines and therapeutics. Supplementary Fig. S2 shows a representative section of sharpened versions of the ensemble map (EMDB entry EMD-11328) compared with EMDB entry EMD-21375, in which the glycans can now be better traced. However, we should not forget that the ensemble map contains images in which the receptor-binding domain (RBD) and N-terminal domain (NTD) are in different positions (see Section 3.2), and consequently these domains appear to be blurred. Details of how the tracing was performed can be found in Section 2, while in Supplementary Fig. S3 we present two map-to-model quality figures indicating the good fit in general, with the obvious exception of the variable parts.