Structural Design and Assessing of Recombinantly Expressed African Swine Fever Virus p72 Trimer in Saccharomyces cerevisiae

Figures

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  Scheme 1. Flow chart of p72 rational design.

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  Figure 1. The target substitution sites for African Swine Fever Virus (ASFV) p72 trimer stabilization. (A) p72 relative B-factor/residue plotted against amino acid residue number. Each secondary structure element with elevated relative B-factor is marked with a circle. Red labels denote residues initially chosen for mutation and black label denotes already exists proline. (B) The side view of the trimeric ASFV p72 (PDB ID: 6KU9) ribbon diagram. One of the p72 is shown in green. Residues within β turn and α helix with elevated relative B-factor are shown as spheres. The finally determined substitution sites are shown as red spheres. The residue located on the most exposed top of the screw propeller-like cap and the already existed proline are shown as orange sphere and green sphere, respectively.

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  Figure 2. Preparation of the purified p72 sample. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified p72, the arrow indicates the band of p72. (B) The Cryo-EM image of p72 trimer particles.

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  Figure 3. Electron microscopic observation results of p72 trimer. (A) Cryo-EM map of the yeast expressed p72 trimer. The map is visualized using UCSF Chimera at a contour level of 0.015. (B) The fitted p72 trimers (red ribbons, 6KU9; blue ribbons, 6L2T) in the cryo-EM map of the yeast expressed p72 trimer. The map is shown as semi-transparent solid surface. The model-map correlation coefficients (CC) are shown upon the structures. (C) The fitted p72 monomer in the cryo-EM map of HeptaPro. Dashed line frame highlights the disordered regions, including BC-CC hairpin, FGN loop and N-terminal base. (D–F) Fit of the p72 backbone into the cryo-EM density for the β sheets and the α helix of N-terminal jelly roll (D), C-terminal jelly roll (E), and crown domain (F).

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  Figure 4. Comparison of the double jelly roll viral capsid or scaffold proteins. (A) The topology of a double jell-roll is shown, all insertions are left out. The β strands and β hairpin loops are colored blue and yellow, respectively. (B) The relative B-factor/residue plot against amino acid residue number of the double jelly roll viral capsid proteins and scaffold protein, including the ASFV p72 (PDB ID: 6KU9), adenovirus hexon (PDB ID: 6B1T), faustovirus MCP (PDB ID: 5J7O), Vaccinia virus scaffold protein D13 (PDB ID: 2YGC), Paramecium bursaria chlorella virus (PBCV) p54 (PDB ID: 1M4X), Sputnik V20 (PDB ID: 3J26), Sulfolobus turreted icosahedral virus (STIV) MCP (PDB ID: 3J31). The secondary structure region corresponding to double jelly roll is highlighted following the color rule in (A). Secondary structure element with elevated relative B-factor is indicated with red triangle marks. Detailed information of each double jelly roll viral capsid protein and scaffold protein is shown in (C).