Table 1.
X-ray data collection, molecular replacement, and refinement statistics. Crystals were grown from a cubic lipid phase (27) using octylglucoside-purified NpSRII (28.0 mg/ml) preincubated with H. salinarum polar lipids (11.2 mg/ml) (11). Ten-microliter aliquots were mixed with 10 μl of monoolein glyceride (Nu-Check Prep., MI) and centrifuged for 1 hour at 11,000g at 22°C. After overnight incubation at 22°C, precipitant was added. The crystals used were thin rods about 5 μm by 20 μm by 200 μm and were examined 5 weeks after the addition of 50 μl of 3.5 M KCl in 50 mM MES (pH 5.3) as precipitant. Addition of exogenous H. salinarum polar lipids was essential for crystal formation. The crystals belong to space group C2221, with a = 87.34 Å, b = 130.81 Å, and c = 50.87 Å, and consist of bilayers stacked in the b direction. The a and c cell dimensions, as well as the packing, are very similar to those reported for 2D crystals of NpSRII (11). Diffraction data were collected on one cryocooled crystal at the microfocus beamline ID13 at ESRF (Grenoble), using a MAR Research charge-coupled device detector. Each image was 1° in φ, with an exposure time of 2 s. Images were reduced, scaled, and merged with the programs DENZO/SCALEPACK (28). Molecular replacement was carried out with the program X-PLOR (29), using the coordinates of the 1.55 Å bacteriorhodopsin structure (PDB code 1C3W, protein without retinal) and yielded an initial R factor of 49.2% (4 to 12 Å). Successive rounds of refinement and model building with the programs CNS (30) and CHAIN (31) using annealed simulated omit and 3|Fo| – 2|Fc| maps resulted in an R factor of 23.3%, and an Rfree of 28.0% for all data between 2.4 and 20 Å without σ cutoff. In many regions near the hydrophobic protein surface, long tubes of density are present for native dihydrophytyl lipids, which as in the case of BR remained tightly bound to the protein through solubilization and cubic lipid phase crystallization. All peptide bonds fall into the allowed regions of the Ramachandran plot.
| Data reduction resolution range | 2.4–20.0 Å | 2.40–2.44 Å |
|---|---|---|
| Total observations | 80,761 | |
| Unique structure factors | 10,704 | 520 |
| Rmerge (%) | 9.5 | 44.0 |
| Average I/σ(I) | 5.7 | 1.3 |
| Completeness (%) | 92.2 | 92.0 |
| Mosaicity (°) | 0.86 | |
| Refinement resolution range | 2.4–20.0 Å | |
| R factor (%) for working set, no σ cutoff | 23.3 | |
| Rfree (%) for 8.7% of the unique structure factors | 28.0 | |
| Deviation from ideal bond lengths (Å) | 0.0086 | |
| Deviation from ideal bond angles (°) | 1.29 |