Abstract P-45: Structure of the Bacteriophage AR9 Bacillus Subtilis Chaperonin According to Cryo-Electron Microscopy

Background: Chaperonins are a family of molecular chaperones Hsp60 (heat shock proteins 60). GroEL is a bacterial chaperonin. It ensures the correct folding of proteins, using the energy of ATP hydrolysis. Three-dimensional reconstructions of its predicted orthologs were obtained and biochemically characterized in free and nucleotide-bound states for bacteriophages EL Pseudomonas aeruginosa, OBP Pseudomonas fluorescens (Kurochkina, L. P. et al., Journal of virology, 2012; Semenyuk, P. I. et al., Biochemical Journal, 2016; Stanishneva-Konovalova, T. B. et al., Journal of Structural Biology, 2020). Physicochemical studies were carried out for the bacteriophage AR9 Bacillus Subtilis and confirmed that the protein has chaperone activity and does not require co-chaperonin to function (Semenyuk P. I. et al., International Journal of Biological Macromolecules, 2020). Methods: The recombinant chaperonin of the B. subtilis bacterial phage AR9 (gp228) was isolated and purified in a free state and vitrified in Vitrobot Mark IV. Data were collected using a Titan Krios cryo-TEM and processed in Warp, RELION and cryoSPARC software. Results: The final structures of the chaperonin were reconstructed with a C1 and C7 symmetry at the resolution of 4.5 Å and 4.0 Å respectively. Significant heterogeneity of the apical domains was addressed further using 3D classification and symmetry expansion in RELION resulting in a set of classes reflecting the conformational transition of the subunits between different states. At least four different conformational states of the subunit were clearly resolved. Conclusion: Gp228 structure show similarities between bacteriophage chaperonin and also bacterial chaperonin GroEL. It is formed by a single ring consisting of seven identical subunits, each has three domains: equatorial, intermediate, and apical. The subunits of the apo-form chaperonin Gp228 exhibit significant conformational flexibility in the apical and intermediate domains

Local Flexibility of a New Single-Ring Chaperonin Encoded by Bacteriophage AR9 Bacillus subtilis

Chaperonins, a family of molecular chaperones, assist protein folding in all domains of life. They are classified into two groups: bacterial variants and those present in endosymbiotic organelles of eukaryotes belong to group I, while group II includes chaperonins from the cytosol of archaea and eukaryotes. Recently, chaperonins of a prospective new group were discovered in giant bacteriophages; however, structures have been determined for only two of them. Here, using cryo-EM, we resolved a structure of a new chaperonin encoded by gene 228 of phage AR9 B. subtilis. This structure has similarities and differences with members of both groups, as well as with other known phage chaperonins, which further proves their diversity

The structure of inactivated mature tick-borne encephalitis virus at 3.0 Å resolution

Tick-borne encephalitis virus (TBEV) causes a severe disease, tick-borne encephalitis (TBE), that has a substantial epidemiological importance for Northern Eurasia. Between 10,000 and 15,000 TBE cases are registered annually despite the availability of effective formaldehyde-inactivated full-virion vaccines due to insufficient vaccination coverage, as well as sporadic cases of vaccine breakthrough. The development of improved vaccines would benefit from the atomic resolution structure of the antigen. Here we report the refined single-particle cryo-electron microscopy (cryo-EM) structure of the inactivated mature TBEV vaccine strain Sofjin–Chumakov (Far-Eastern subtype) at a resolution of 3.0 Å. The increase of the resolution with respect to the previously published structures of TBEV strains Hypr and Kuutsalo-14 (European subtype) was reached due to improvement of the virus sample quality achieved by the optimized preparation methods. All the surface epitopes of TBEV were structurally conserved in the inactivated virions. ELISA studies with monoclonal antibodies supported the hypothesis of TBEV protein shell cross-linking upon inactivation with formaldehyde.</p