# Molecular Organisation of Tick-Borne Encephalitis Virus
## 3 Results
### Lipid Factors  
For each E-M dimer within the asymmetric unit, the reconstruction contained two densities not accounted for by the protein model. These densities are consistent with recently described flaviviral lipid factors [16; 17; 18; 19]. The density quality did not allow us to unambiguously identify the lipid species or fully model the fatty acid tails. As phosphatidylinositol (PC) is the most common lipid species found in flavivirus virions, we built PC headgroups and the first four carbon atoms of each fatty acid tail [47]. The PC are located near the lipid bilayer and closely associate with domain IV of the E protein and the M protein N-terminal loop. PC 1 lies between E-H1 and E-H2 (Figure 5A,B). PC 2 is located between E-H1, E-H2, E-H3, the C-terminus of E-TM2, and the N-terminal loop of M (Figure 5A,B). PISA analysis revealed that the pocket for PC 1 consists of 13-15 E protein residues (File S8). The surface area of the pocket is 313.6, 327.2, and 305.2 A\({}^{2}\) in chains A, B, and C, respectively. The importance of the lipids was assessed with the PISA Complex Formation Significance Score (CSS) on a scale from 0 to 1, where 0 indicates no role in  
Figure 4: Interactions at the E heterotetramer interface. (**A**) Interactions between the E proteins located at the heterotetramer interface. Chain B is shown in pink, chain A in light green and chain C in grey. The asymmetric units (AU) are labelled. Interacting residues are shown as stick representations and labelled in the close-up views. Hydrogen bonds are shown by green dotted lines. The distance between the interacting residues is labelled. The \(\pi\)-stacking interaction between His347 and Arg187 and the salt bridge between Arg187 (atom-NE) and Asp380 are not shown for clarity. His347 and Arg187 are separated by a distance of 3.4 Å, and Arg187 (atom-NE) and Asp380 by 4 Å. (**B**) A consensus sequence of the interacting E residues in 183 TBEV sequences. Letter size is 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). The wave indicates a sequence break. (**C**) Structure-based sequence alignment of flaviviruses with high-resolution structures. Residues conserved throughout flaviviruses are highlighted with pink, and residues conserved in TBEV isolates are highlighted in grey. The interacting residues are numbered according to TBEV Kuutsalo-14 sequence (GenBank: MG589938.1). complex formation and 1 indicates an essential role. CSS scores of 0.404, 0.404, and 0.463 for chains A, B, and C, respectively, indicate an auxiliary role. Additionally, PC binding is energetically favourable with \(\Delta\)G values of \(-4.1\), \(-7.3\), and \(-4.7\) kcal/mol for chains A, B, and C, respectively. The least solvent-accessible pocket residues are Thr406, Gly409, Arg412, Ala420, and Phe423 (File S8). Residues Thr406, Arg412, and Trp421 are within 4 A of the PC 1 (Figure 4C). In chains A and B, Thr406 forms a hydrogen bond with the PC 1 proximal ester. Additionally, Arg412 forms hydrogen bonds with the PC 1 phosphate group and Trp421 interacts with the choline group via a cation-\(\pi\) interaction in all chains [48]. Furthermore, Ile410 is within 4 A of PC 1, and the positive \(\Delta\)G value of the residue indicates that it interacts with the fatty acid tail hydrophobically. Thr406, Arg412, and Trp421 are conserved in all analysed sequences and Ile410 only in TBEV (Files S2, S4, and S7). Four M residues and 18-19 E residues form the PC 2 pocket (File S8). The interaction of PC 2 with the E protein chains A and C was determined to be essential (each CSS score is 0.590), but the interaction with chain B is auxiliary (CSS score 0.350). In contrast, the M protein interaction does not contribute to the complex assembly as the CSS scores were 0 for all M chains.