Article Figures & Data
Figures
- FIG. 1.
(A) Ribbon diagram of the crystal structure of the ZiPD homodimer at a resolution of 2.9 Å. Subunit A is red, subunit B is blue, and the zinc ions are represented by green spheres. (B) Active site cleft 1 of ZiPD homodimer formed jointly by subunits A and B. His248 and His 270, which are essential for cooperativity, are yellow, and the remaining zinc ligands are represented by cyan sticks.
- FIG. 2.
Stereo view of ZiPD zinc coordination site with the experimental electron density map shown at 1σ above the mean. Zinc ions are represented by green spheres. Bond lengths (in angstroms) between Zn2 and His270 and between Zn2 and His248 are indicated to update previous spectroscopic and mutational analyses in which determining which of the two residues coordinates zinc was impossible.
- FIG. 3.
Sequence alignment of tRNase Z-family proteins. ECOLI, E. coli ZiPD; BACSU, B. subtilis tRNase Z; TMAR, T. maritima tRNase Z. Conserved metal coordinating residues are highlighted in blue, and exosite residues are indicated by a yellow background. Secondary structure features are indicated by red arrows for β-strands and by blue cylinders for α-helices. unknown.
- FIG. 4.
Comparison of the structures of tRNase Z-family proteins E. coli ZiPD, B. subtilis tRNase Z (subunits A and B) (8), and T. maritima tRNase Z (11). The RMSD values for the protein cores were calculated to be 1.0 Å for E. coli-B. subtilis (subunit B) using 254 carbon alpha atoms and 2.1 Å for E. coli-T. maritima using 241 carbon alpha atoms. The location of the exosite with respect to the protein core is indicated in the E. coli ZiPD diagram by dotted lines.
- FIG. 5.
Superposition of ZiPD and B. subtilis tRNase Z carbon alpha chains. ZiPD is blue, and tRNase Z is yellow. The RMSD value between the exosites was calculated to be 1.5 Å using the carbon alpha atoms of 51 residues.
- FIG. 6.
Comparison of active sites with and without PO4 bound from E. coli ZiPD (A) and B. subtilis tRNase Z subunit B (bound to PO4) (B). Hydrogen bonds to the phosphate molecule are magenta and highlight the key role of His248 and His270 in determining substrate orientation.
Tables
- TABLE 1.
Summary of data collection statistics for multiple anomalous diffraction and native data sets of ZiPD
Parameter Inflection Peak Remote Native Beam line BW7Aa BW7A BW7A X06SAb Wavelength (Å) 1.282 1.284 1.129 1.000 Resolution (Å) 20 to 3.0 20 to 3.0 20 to 3.0 50 to 2.9 No. of reflections 109,441 49,383 124,515 312,291 No. of unique reflections 17,927 16,989 18,160 20,193 Completeness (%) 99.5 (100)c 93.8 (97.7) 99.5 (99.9) 99.8 (99.9) I/σ (I) 22.7 (2.7) 17.6 (1.5) 27.1 (1.8) 23.4 (3.6) R merge d 0.067 (0.79) 0.057 (0.66) 0.049 (0.88) 0.071 (0.75) ↵ a EMBL Hamburg Outstation, DESY.
↵ b Swiss Light Source at Paul Scherrer Institut.
↵ c The numbers in parentheses correspond to the last resolution shell.
↵ d R merge(I) = $$mathtex$$\({{\sum}_{\mathit{hkl}}}{{\sum}_{\mathit{i}}}{\,}{\vert}{\,}\mathit{I_{i}}(\mathit{hkl}){-}\overline{\mathit{I}(\mathit{hkl})}{\,}{\vert}{\,}/{{\sum}_{\mathit{hkl}}}{{\sum}_{\mathit{i}}}\mathit{I_{i}}(\mathit{hkl}).\)$$mathtex$$
- TABLE 2.
Summary of refinement statistics for the native ZiPD data at a resolution of 2.9 Å
Parameter Value Unit cell a = 147.2, b = 147.2, c = 138.2 Space group P6422 No. of protein residues 305 No. of protein atoms 2,268 Resolution range (Å) 50 to 2.9 No. of observations (total) 20,037 No. of observations for Rwork 19,099 No. of observations for Rfree 974 R work 0.23 R free 0.25 RMSD for bond lengths (Å) 0.01 RMSD for bond angles (Å) 1.6
