Table 1.
Summary of crystallographic analysis
| Native | SeMet (peak) | |
|---|---|---|
| Data collection | ||
| Wavelength (Å) | 1.075 | 0.9791 |
| Space group | R32 | R32 |
| Cell dimensions (Å) | ||
| a, b, c (Å) | 142.8, 142.8, 187.8 | 142.4, 142.4, 187.7 |
| α, β, γ (°) | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 |
| Resolution (Å) | 50.0-2.30 (2.34-2.30) | 50.0-2.54 (2.59-2.54) |
| Rsym (%) | 6.8 (84.0) | 10.2 (87.4) |
| I/σ̣I | 45.1 (4.8) | 28.8 (4.1) |
| Completeness (%) | 99.9 (100.0) | 100.0 (100.0) |
| Redundancy | 21.8 (21.3) | 22.3 (22.4) |
| SAD Analysis | ||
| Rms FH/ε | 0.85 | |
| Mean FOM before DM | 0.43 | |
| Mean FOM after DM | 0.64 | |
| Refinement | ||
| Resolution (Å) | 50.0–2.30 | |
| No. of reflections (|F|>0σ) | 31,175 | |
| R-factor/Rfree | 21.6/23.4 | |
| Total protein atoms | 2,510 | |
| Water molecules | 178 | |
| B-factors (Å2) | ||
| Protein | 31.64 | |
| Water | 39.15 | |
| R.m.s deviations | ||
| Bond lengths (Å) | 0.007 | |
| Bond angles (°) | 1.161 | |
| Ramachandran (%) | ||
| Within favored | 98.0 | |
| Within allowed | 99.8 | |
| Outliers | 2 (Gln285 and Lys294) | |
The detailed experimental procedures are provided in SI Materials and Methods. In brief, recombinant B. subtilis GerBC (residues 25–374) was expressed as a His6 fusion protein in Escherichia coli. The GerBC protein was crystallized from a solution consisting of 1.5–1.7 M ammonium sulfate and 0.1 M sodium acetate (pH 4.6) by the hanging-drop vapor diffusion method at 4°C. The structure of GerBC was determined by the single-wavelength anomalous dispersion (SAD) method using data from a selenomethionine-substituted crystal.
Values in parentheses are for the highest-resolution shell. Rsym = ΣhΣj |Ih,j−Ih|/ΣhΣj Ih,j for the intensity (I) of i observations of reflection h. Rms FH/ε = (1/nΣFH2)1/2/(1/nΣε2)1/2, where FH is the structure factor amplitude for anomalous scatterers and ε is the residual lack of closure error. Figure of Merit (FOM) = <ΣP(α) exp(iα)/ΣP(α)>, where P(α) is the probability distribution for the phase. FOM before DM indicates the figure of merit before density modification. R-factor = Σ‖Fobs|−|Fcalc‖/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree = R-factor calculated using 5% of the reflection data chosen randomly and omitted from the start of refinement.