# Molecular Organisation of Tick-Borne Encephalitis Virus
## 3 Results
### Quasi-Equivalence within the Asymmetric Unit  
The three E and M monomers within the asymmetric unit occupy quasi-equivalent positions (Figure 2B). Superimposition of these monomers indicates that conformational differences in the peripheral and transmembrane helices, along with the different interfaces provided by the heterotetramer ectodomain packing described previously, account for these differences [4]. The E monomers exhibit a RMSD of 1 A, whereas the M monomers have a  
Figure 2: Quasi-equivalence within the asymmetric unit. **(A)** Surface representation of the TBEV virion. One asymmetric unit is highlighted. The symmetry axes are indicated with an ellipse (two-fold), a triangle (three-fold), and stars (five-fold). **(A–C)** The three E proteins forming the asymmetric unit are coloured in light green (chain A around the five-fold axes of symmetry), pink (chain B around the three-folds), and grey (chain C around the two-folds). **(B)** Cartoon representation of the E and M membrane-associated domains within the context of a virion. **(B–F)** The three M proteins within one asymmetric unit are coloured in purple (chain D), magenta (chain E), and turquoise (chain F). **(C)** Superimposition of the three E–M heterodimers that make up the asymmetric unit, based on the E proteins. Transmembrane helices of E are labelled as E–TM1, and E–TM2; perimembrane helices are labelled E–H1, E–H2, and E–H3. **(D)** Detailed view of the superimposed M proteins of one asymmetric unit. Transmembrane helices of M are labelled as M–TM1, and M–TM2; perimembrane helix is labelled M–H1; N-terminal loop and hinge are indicated. **(E)** Superimposition of the three M proteins in one asymmetric unit as in **(C)**. The hinge between M–H1 and the N-terminal loop is highlighted in yellow and is shown zoomed in. The differences in the backbone positions at residues 23 and 24 are indicated. **(F)** Superimposition of the three M proteins in one asymmetric unit as in **(C)** with the N-terminal residues 1–21 hidden for clarity. The angles between the superimposed helices are shown in the top view (left), and the distances between the corresponding C\(\alpha\) atoms are indicated in the side view (right).  
higher RMSD of 1.7 A over all C\(\alpha\) atoms. Superimposition of the three E-M heterodimers within the asymmetric unit, based on the E protein ectodomains, indicated conformational differences between the M proteins located on the two-fold (chain D), three-fold (chain E), and five-fold (chain F) symmetry axes (Figure 2). Chain E has a C\(\alpha\) RMSD of 1.8 A to chain D, and of 1.4 A to chain F; however, chains D and F have a smaller C\(\alpha\) RMSD of 0.8 A (Figure 2). The N-terminal loop regions (residues 1-24) and the C-terminal helical regions (residues 25-74) are similar between all M proteins, with low RMSD values of 0.4 A and 0.5 A, respectively.  
The major conformational differences between the three M proteins arise due to the different locations of Asp23 and Ser24 that diverge by up to 2.8 A at the C\(\alpha\). These residues form a hinge immediately preceding the M-H1 helix, and are highly conserved in all analysed TBEV isolates (Files S3 and S5, Table S2, Figure S5). In comparison to chain E, the M-H1 helices are rotated anticlockwise in chains D (18\({}^{\circ}\)) and F (15.6\({}^{\circ}\)) (Figure 2). In addition, the three M-TM1 helices pack at different tilt angles relative to the E transmembrane helices. The major difference occurs at the N termini of the helices where the relative positions in chains D and F differ from chain E by 7.8 A (Figure 2D,F).