# Molecular Organisation of Tick-Borne Encephalitis Virus
## 3 Results  
### Cryo-EM Reconstruction of the UV-Inactivated TBEV Viron  
The purified TBEV was subjected to UV-C irradiation at an energy of 0.1 J/cm, causing the virus titre to drop from 3 \(\pm\) 2 \(\times\) 10\({}^{10}\) pfu/mL to below the level detectable by plaque assay (50 pfu/mL) (average of three repeats). Gel electrophoresis confirmed that the structural proteins were not cross-linked during this process (Figure S1). Cryo-electron micrographs of the UV-inactivated virus showed intact spherical virions \(\sim\)50 nm in diameter along with some broken particles (Figure S2A). Three datasets were collected at 300 keV and reconstructed. An initial small dataset was collected on a K2 detector and reconstructed to a resolution of 3.5 A (preparation 1). Two larger datasets were collected on a K3 detector, reconstructed independently to 3.7 A and 3.2 A (preparations 2 and 3, respectively). There were no significant differences between the three reconstructions. No density could be attributed to the presence of the isoxazole compounds included in the sample preparation, nor were there conformational changes in E that would accommodate the compounds. The two K3 datasets were combined, and on reclassification, did not split into two separate classes based on sample preparation, indicating their similarity. The merged K3 datasets gave a final reconstruction with a resolution of 3.3 A (Figures 1 and S2B,C). The final reconstruction had the clearest sidechain densities and was used to build the E and M atomic models (Figures 1 and 2, Table S1). The ectodomains and the envelope leaflets were well resolved, the peripheral membrane helices and the transmembrane helices were slightly worse, but no features were distinguished in the NC (Figures 1, 2 and S3). Our virion reconstruction is consistent with the previously published TBEV structure of a different strain but is at a higher resolution, allowing the visualisation of side chains and their interactions [4]. We therefore conclude that UV inactivation did not cause recognisable damage to the virion structure. Figure 1: _Cont._  
Figure 1: TBE virion cryoEM reconstruction and atomic model. (**A**) Radially coloured isosurface representation of the reconstruction viewed down a two-fold axis of symmetry, shown at 2.35 \(\sigma\) above the mean. The symmetry axes are indicated with an ellipse (two-fold), a triangle (three-fold), and star (five-fold). The colour key is the same as in panel B. (**B**) A 60 Å central slice of a radially coloured isosurface representation of the reconstruction viewed down a two-fold axis of symmetry, at 2.35 \(\sigma\) above the mean. (**C**) Surface representation of the reconstruction, with a raft of three E–M–M–E heterotetramers highlighted, viewed down a two-fold axis of symmetry. (**D**) Cartoon representation of the E–M–M–E heterotetramer shown from the top and side views. (**C**,**D**) The E domains are coloured in purple (domain I), turquoise (domain II), magenta (domain III), grey (domain IV), the E fusion loop is coloured in gold, the E Asn154 glycan motif in black, the M protein in green, and the phosphatidylcholine (PC) lipids in red-orange. Figure 2: _Cont._  
The surface of the virion is composed of 30 raft-like structures, each containing three E-M-M-E heterotetramers. Each heterotetramer consists of two antiparallel E-M heterodimers (Figure 1). The asymmetric unit contains three E-M heterodimers (Figure 2). The root-mean-squared deviation (RMSD) between the current model and the published 3.9 A model of Hypr strain is 1.6 A for the corresponding C\(\alpha\) atoms [4]. The two distinguished envelope leaflets follow the polygonal shape typical of flaviviruses and the bilayer is constricted in the areas where the E and M transmembrane helices insert (Figure 1) [4; 41]. The E protein is divided into four domains: three primarily \(\beta\)-sheet domains (domains I-III) and an \(\alpha\)-helical domain (IV) (Figure 1). Domain II contains a hydrophobic fusion peptide at its tip (residues 100-109) [4; 12].