Figure 1.
Double-stranded DNA binding of S. pyogenes Csn2.
A: Sequences of S. pyogenes CRISPR and control DNA fragments used for an electrophoretic mobility shift assay. The repeat and the first spacer of S. pyogenes CRISPR are shown in red and green, respectively. The control DNA fragment contains the promoter site of the Early Responsive to Dehydration Stress 1 gene from A. thaliana. B: An electrophoretic mobility shift assay was performed with 150 ng of dsDNA (90 bp) and increasing concentrations of S. pyogenes Csn2. The molar ratio of DNA to S. pyogenes Csn2 tetramer is indicated for each lane.
Table 1.
Data collection and refinement statistics.
Figure 2.
Sequence alignment and monomeric structure of S. pyogenes Csn2.
A: Sequence alignment of Csn2 homologues from S. pyogenes, E. faecalis and S. thermophilus. Secondary structure elements are indicated based on the S. pyogenes Csn2 structure. The calcium coordinating residues within the CA1 and CA2 sites are marked with orange and purple triangles, respectively. B: Structure of S. pyogenes Csn2 monomer A. The globular α/β domain, the extended α-helical domain and the hinge regions are shown in green, cyan and yellow, respectively. Secondary structure elements are also indicated. Bound calcium ions in the CA1 and CA2 sites are represented as orange and purple spheres, respectively.
Figure 3.
Tetrameric structure of S. pyogenes Csn2.
A: Tetrameric arrangement of S. pyogenes Csn2. Monomer A is colored as in Figure 2B, and monomer B is shown in pink. The tetramer is viewed along the two-fold symmetry axis. The two S. pyogenes Csn2 monomers found in the asymmetric unit are enclosed by the black dashed line. B: Analytical size-exclusion chromatography of S. pyogenes Csn2. Elution profiles with different buffer conditions are represented by different colors. Elution volumes for molecular weight standards are also indicated. C: Electrostatic potential surface (red = −25 kT, blue = +25 kT) of the S. pyogenes Csn2 tetramer. Pymol (www.pymol.org) was used to calculate APBS electrostatics including the bound calcium ions [27].
Table 2.
Concentration (ppb) of metals in S. pyogenes Csn2 samples.
Table 3.
Comparison of S. pyogenes Csn2 structure with the metal identified as calcium, sodium, potassium or water.
Figure 4.
Calcium binding in S. pyogenes Csn2 structure.
The CA1 (A) and CA2 (B) sites are colored as in Figure 2B, and coordinating oxygen atoms are shown in red. Among the two CA1 sites, the one adjacent to monomer B, which has lower B factors, is shown. The missing water molecule in the CA1 site, represented as a blue sphere, is modeled based on a comparison with the other CA1 site. Asp122 is shown in its calcium-binding conformation. The difference electron density map for the calcium ions was contoured at 10σ. The distances between the calcium ions and the coordinating atoms are also indicated.
Figure 5.
Conformational changes in S. pyogenes Csn2 structure.
A: Structural alignment of S. pyogenes Csn2 monomers based on their α/β domains. Cα traces of the two monomers are colored as in Figure 2B except for the calcium ions in the CA1 sites of monomers A and B, which are shown in cyan and pink, respectively. B: Structural alignment of S. pyogenes and E. faecalis Csn2 tetramers based on their α/β domains. S. pyogenes and E. faecalis Csn2 tetramers are shown in red and blue, respectively. The two-fold symmetry axis is also indicated.