Figure 1.
Characterization of Cthe_2751 (A) Sequence alignment of Cthe_2751 homologues.
Only the top 11 matches with Cthe_2751 amino acids 1–134 are shown. Conservation is colored according to ClustalW convention. (B) Size exclusion profile of Cthe_2751 run on Hi Load 10/300 Superdex G75 gel filtration column equilibrated with 20 mM Tris-HCl, 100 mM NaCl, pH 8.0, reveals that the protein exists as a dimer in solution. SDS-PAGE picture (inset) showing the purity of Cthe_2751 before crystallization. (C) Sedimentation velocity experiments performed using an analytical ultracentrifuge suggested that Cthe_2751 forms a dimer in solution. The curve was generated using Sedfit software.
Figure 2.
Structure of singleton Cthe_2751.
(A) Topology diagram of the structure. (B) Cartoon representation of Cthe_2751 with helices shown as cylinders in (C) to depict the contour, pairing and stacking. (D) Cartoon representation of a dimer of Cthe_2751.
Figure 3.
Two dimensional projection (along C axis direction) of 2-fold symmetry related molecules for space group P4122 and C2221.
The transformation between space groups P4122 and C2221 is illustrated. (A) The projection of Cthe_2751 monomer (dark blue) and 7 symmetry related molecules along C axis. The homodimer of Cthe_2751 (dark and light blue molecules) is related by crystallographic 2-fold 2(x 0 0). Please note that 41 screw symmetry related molecules are not shown for the sake of only displaying the transformation between P4122 and C2221 space groups; (B) In order to illustrate the transformation between P4122 and C2221 space groups, Figure 3A is rotated 45 degrees clockwise around 41 axis; (C) The projection of Cthe_2751 dimer along C axis. The 4 Cthe_2751 dimers in C2221 space group have almost the same orientation as that of the 8 Cthe_2751 monomers (or 4 dimers) in the 45° rotated P4122 unit cell.
Figure 4.
The transformation between space groups P4122 and C2221.
(A) The projection of symmetry elements in space group P4122 along 41 axis; (B) Degeneration of P4122: 41 axis are transformed into 21 axis, while the 2 fold axis, generated by 41 axis, disappear too. The cell is rotated clockwise by 45° around 41 axis; (C) The cell parameter a′ and b′ in C2221 space group take the diagonal direction along a+b and a−b of space group P4122, respectively, and forms new unit cell; (D) In order to follow the international conversion of space group C2221, the origin is translated 3/8 cell length along c axis.
Table 1.
Statistics of inter-dimer distance between 2 neighboring dimers in the 3 space groups.
Figure 5.
(A) The side chain of Tyr88 of chain A protrudes into a concave cavity formed by Leu52, Pro53, Leu84 and Tyr88 of chain B. (B) Chain A shown in surface representation, while chain B is depicted as a cartoon. (C) Representative 2Fo-Fc electron density for some of the residues at the dimer interface contoured at 1.0 σ.
Table 2.
Inter monomer hydrogen bonds identified by PISA analysis of the Cthe_2751 dimer.
Figure 6.
(A) Cartoon of the model predicted by I-TASSER (B) Superposition of the Cα atoms of the predicted structure (magenta) over the experimental crystal structure (blue). (C) Average distance tree for Cthe_2751 constructed by the Jalview 2.6.1 Java alignment editor using BLOSUM62.
Table 3.
Details of models built by different 3D structure prediction programs.
Figure 7.
Functional analysis of Cthe_2751.
(A) Cluster of aromatic and charged residues of Cthe_2751. N and C terminals are marked; Cthe_2751 is depicted as a cartoon, amino acids as sticks. (B) Nucleic acid binding ability of Cthe_2751 was tested in an EMSA assay. Cthe_2751 could not bind double stranded nucleic acids similar to bovine serum albumin (BSA) under the assay conditions. Lambda repressor protein was used as a positive control.
Table 4.
Data collection and refinement statistics.