Article Figures & Data
Figures
- FIG. 1.
Online multiangle laser light-scattering, absorbance, and refractive index analysis of ORF12 in solution. The abscissa indicates the time of elution from the high-pressure liquid chromatography column; the left ordinate indicates the molar mass in g/mol (Da). The absorption peaks are in blue (native) and red (SeMet), and the dashed lines indicate the molar masses. The experimental masses are given in black (kDa). The native ORF12 has been injected at a concentration of 6 mg/ml, and the SeMet derivative has been injected at 8 mg/ml.
- FIG. 2.
Structure of p2 ORF12. (A) Crystallographic trimer ribbon representation with monomers identified by their letter in the PDB (3D8L). The ribbon is colored blue to red from the N to the C terminus. The surface interaction areas between each monomer are given. (B) Stereo view of ORF12 monomer, with the same coloring as in panel A. The helices are numbered 1 to 5.
- FIG. 3.
View of the spirals formed by ORF12 crystal packing. The crystal is formed from such spirals packed side by side. (A) Side view of the two spirals side by side. Each monomer is colored brown, violet, and yellow repeatedly. (B) View of the spirals rotated by 90° around the vertical axis. The gross dimensions of the spiral are displayed. (C) Stick representation of a stretch of the two side-by-side-positioned spirals illustrating the dense network of charge interactions, but loose packing, between the two spirals. The color code is blue for positively charged residues, red for negatively charged residues, pale green for semipolar residues, green for methionines, yellow for aliphatics, and violet for aromatics.
- FIG. 4.
Intracellular detection of ORF12 during phage p2 infection of L. lactis MG1363. Samples were taken at various time intervals after phage infection, and ORF12 was detected by Western blot analysis. For ORF12, 5 ng of purified protein and, for p2, 1 × 1010 PFU of CsCl-purified phages were loaded, which corresponded to 7.8 ± 1.5 μg.
- FIG. 5.
Model of complex between the TMP hydrophobic helix and ORF12 spiral. (A) Sphere representation of a segment of four ORF12 modules in the spiral. The hydrophobic patches are visible in the center of the twisted spiral, in a twisted crevice, formed of aromatic residues. The color code is blue for positively charged residues, red for negatively charged residues, pale green for semipolar residues, green for methionines, yellow for aliphatics, and violet for aromatics. (B) View of the TMP segment 777 to 818, modeled as a curved α-helix. All atoms are colored orange, and the aromatic side chains are colored pink. The periodicity of the aromatic residues of TMP coincides with that observed in the ORF12 spiral, as outlined by the red arrows. (C) Model of a complex between the TMP segment 777 to 818 and four ORF12 modules in the spiral. Color coding is the same as for panels A and B. (D) Sequence of p2 TMP (1 to 999). The repeat area (522 to 898) is underlined. The segment chosen in the above model is identified in a yellow box with aromatic residues shown in violet.
Tables
- TABLE 1.
Data collection and refinement statistics for p2 ORF12
Parameter Valuea Data collection PDB access code 3D8L Space group R 3 2 Unit cell (Å) 158.3 158.3 99.5 90.0 90.0 120.0 Beamline ID14-EH4 Detector ADSC Q315 Wavelength (Å) 0.9785 Rotation range (°) 360 Resolution range (Å) 35.0-2.90 (3.06-2.90) No. of observations 230,325 (31,122) No. of unique reflections 10,739 (1,556) Completeness 99.9 (100) Redundancy 21.4 (20.0) I/sI 29.1 (6.4) R sym (%) 9.2 (44.1) Refinement Resolution range (Å) 30.0-2.9 (2.98-2.90) No. of unique reflections 10,218 No. of atoms of protein per water/buffer 22,114 R/R free all (last shell) 25.3/20.3 (33.5/29.4) RMSD Bonds (Å) 0.015 Angles (°) 1.591 Mean B value (Å2) Protein 64.43 Water 51.33 Ramachandran (%) Favored region 92.8 Allowed regions 5.7 Generously allowed regions 1.5 ↵ a All values in parentheses belong to the last shell.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Fig. S1, view of a segment of the surface of ORF12.
Zipped TIF file, 89K. - Supplemental file 2
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Fig. S2, stereo view of a model complex of a segment of the ORF12 surface.
Zipped TIF file, 211K. - Supplemental file 3
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Legends to Fig. S1 and S2.
MS Word document, 22K.
- Supplemental file 1
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Fig. S1, view of a segment of the surface of ORF12.
