VOLUME 291 (2016) PAGES 3668–3681
Based on the reprocessed diffraction data for Rv2837c crystal, a better model and electron density was obtained at a little higher resolution of 2.3 Å (Table 1). The new electron density shows that the two protein molecules in the asymmetric unit are in different states. In molecule A, the Fo − Fc map clearly indicates the existence of a catalytic water molecule between the two Mn2+ ions and pApA-like molecule. In molecule B, water molecule is not observed, and the Fo − Fc map suggests two AMPs (Fig. 10). As a precaution, both pApA and AMP were fitted in the active site of each protein molecule and refined. The result clearly shows that two AMPs should be predominant in molecule B and that a pApA fits into the electron density better than two AMPs in molecule A. It also indicates that AMPs are not compatible with the catalytic water molecule. Consequently, we hypothesize that even though molecule A contains a mixture of pApA and AMP, pApA still outweighs AMP (Fig. 11). Thus, in the final model, a pApA is placed in the active site of molecule A, and two AMPs are placed in the active site of molecule B. That means our structure not only catches a snapshot in the middle of catalysis, but it also shows the last moment of the catalysis before the product leaves the active site. It is also confirmed that the new structure is still supportive to the mechanism previously proposed in the paper.
TABLE 1.
X-ray diffraction data collection and refinement statistics
| Rv2837c-C/pApA, AMP | |
|---|---|
| Data collection | |
| Wavelength (Å) | 0.9791 |
| Space group | P212121 |
| Unit cell parameters | |
| a, b, and c | 58.4, 99.3, and 108.7 Å |
| α, β, and γ | 90.0, 90.0, and 90.0° |
| Resolution (Å)a | 50–2.30 (2.38–2.30) |
| Total no. of reflections | 129,750 |
| No. of unique reflections | 26,386 |
| Completeness (%)a | 93.8 (99.4) |
| Redundancy a | 4.9 (5.6) |
| I/σ 〈I〉a | 35.77 (16.95) |
| Rmerge (%)b | 8.4 (19.2) |
| Refinement | |
| Rwork/Rfree (%)c | 21.70/28.90 |
| No. atoms | |
| Protein | 4801 |
| 5′-pApA | 44 |
| 5′-AMP | 46 |
| Mn2+ | 4 |
| Water | 166 |
| Average B-factors (Å2) | |
| Protein | 34.72 |
| 5′-pApA | 48.28 |
| 5′-AMP | 42.48 |
| Mn2+ | 26.66 |
| Water | 37.88 |
| Root mean square deviations | |
| Bond lengths (Å) | 0.008 |
| Bond angles | 1.037° |
| Ramachandran plot (%) | |
| Favored | 97.00 |
| Allowed | 3.00 |
| Generally allowed | 0 |
a Values in parentheses refer to the high resolution shell.
b Rmerge = Σ Σ|I(k) − 〈I〉|/Σ I(k) where I(k) and 〈I〉 represent the diffraction intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements.
c Rwork = Σ‖Fobs| − k|Fcalc‖/|Fobs| where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree is the sum extended over a subset of reflections (5%) excluded from all stages of the refinement.
FIGURE 10.
The pApA and AMP were placed in the active sites of Rv2837c dimer according to the Fo − Fc map that is contoured at the 3σ and 2σ level, respectively. A, stereoview of the pApA at the active site of molecule A. B, stereoview of two AMPs at the active site of molecule A. C, stereoview of two AMPs at the active site of molecule B. The Fo − Fc map is contoured at 3σ level in the upper lane, and the lower lane is at 2σ level. Substrates are shown as sticks; the Mn2+ ions are denoted by magenta spheres, whereas the catalytic water molecule is shown as a red sphere. The 3′-5′ bond of pApA was clearly found in molecule A, but it was not evident in molecule B when the Fo − Fc map is contoured at 2σ level.
FIGURE 11.
Electron density for pApA and AMP of 2Fo − Fc map contoured at the 1.0σ level. A, stereoview of pApA at the active site of molecule A. B, stereoview of two AMPs at the active site of molecule A. C, stereoview of two AMPs at the active site of molecule B. pApA and AMP are shown as sticks; the Mn2+ ions are denoted by magenta spheres, whereas the catalytic water molecule is shown as a red sphere.
The atomic coordinates and structure factors (code 5JJU; Rv2837c-pApA/AMP) have been deposited in the Protein Data Bank (http://wwpdb.org/).