# Molecular Organisation of Tick-Borne Encephalitis Virus
## 3 Results
### Stabilising the Raft Structure--Interactions between the Heterotetramer Complexes  
Previous studies on flavivirus virion structures have focused on the interactions within a single E-M-M-E heterotetramer, whereas here we describe new inter-heterotetramer interactions of the membrane regions. Chains D and F twist towards each other and interact at the heterotetramer interfaces within the raft (Figures 2B, 3A and S4). Each interface has a buried surface area of 173.2 A\({}^{2}\) and a free energy gain upon interface formation (\(\Delta\)G) of 1.1 kcal/mol. The interaction predominantly occurs between the two M-H1 helices, which are separated by a distance of 19.1 A (Table S2). One \(\pi\)-stacking interaction between Trp37 on each M protein, and two salt bridges between Lys40 and Glu33 stabilise the M protein interface (Figure 3A). Figure 3: Interactions at the M heterotetramer interface. (**A**) Interaction between the two tilted M proteins located at the heterotetramer interface. Chain D is shown in purple and chain F shown in turquoise. Interacting residues are shown as stick representations and labelled in the close-up views (right). Hydrogen bonds are shown by green dotted lines. The distance between the interacting residues is labelled. (**B**) Sequence conservation of the interacting M residues in 183 TBEV isolates. The consensus residues are shown with the size of the letter proportional to the frequency of the residue. Interacting residues are marked with numbers and the residues are coloured according to chemistry (grey, hydrophobic; pink, acidic; purple, polar including glycine; turquoise, basic; magenta, neutral). (**C**) Structure-based sequence alignment of flavivirus. Positions conserved throughout flaviviruses are highlighted in pink, and positions conserved only in the majority of TBEV strains are highlighted in grey. The interacting residues are numbered according to TBEV Kuutsalo-14 sequence (GenBank: MG589938.1). Multiple sequence alignment of the M proteins of 183 TBEV isolates belonging to all three TBEV subtypes revealed that Trp37 and Glu33 are conserved across all analysed sequences. In addition, Lys40 is conserved in 173 of the analysed TBEV isolates. The remaining 10 isolates, including the Hypr strain, contain an arginine residue at position 40 instead (Figure 3B, Files S3 and S5). Although arginine can also function as a hydrogen donor, no interaction between Arg40 and Glu33 is observed in the previously published Hypr strain structure [4]. Furthermore, the M proteins of Hypr do not interact at the heetrottramer interface as the M-H1 helices are separated by a larger distance of 25.7 A (Table S2, Figure S4) [4]. Analysis of all other available flavivirus structures with a resolution higher than 4 A revealed that the M-H1 helices are separated by distances ranging from 28.9 A to 38.6 A (Table S2, Figure S4) [4; 17; 18; 19; 42; 43; 44; 45; 46]. Consequently, none of the previous structures contain M protein interactions at the inter-heterotetramer interfaces. Structure-based sequence alignment of M proteins shows that Glu33 is conserved in insect-borne flaviviruses (Figure 3C). However, Trp37 and Lys40 are not conserved (Figure 3C, File S1). This suggests that the interaction can only form in tick-borne flaviviruses (TBFVs) containing the three interacting residues. Interactions at the intra-raft heetrottramer boundary also occur between the E proteins (Figure 4, File S6). The total buried surface area at the E protein interface is 1095 A\({}^{2}\). The \(\Delta\)G is \(-8\) kcal/mol, of which \(-4.8\) kcal/mol arises from hydrophobic interactions at the domain II-domain II (DII-DII) interface. At the interface, there are two \(\pi\)-stacking interactions between His229 of one chain and the Pro79 of the other (Figure 4A,B). In addition, we identify seven pairs of interacting residues at the domain I-domain III (DI-DIII) interfaces (Figure 4B, File S6).