The DppA protein was expressed in E. coli BL21 Gold and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. A final concentration of 8 mg ml−1 was used for crystallization experiments.
Crystals of DppA protein could be obtained with several precipitants from the initial screen using the vapour-diffusion method. The crystallization conditions were further optimized and tiny needles that were too small for data collection appeared after several days in most cases (0.1 M sodium acetate pH 4.6, 28–31% PEG 400, 0.1 M MgCl2; 0.1 M sodium phosphate–citrate pH 4.2, 0.2 M K2HPO4, 1.6 M NaH2PO4; 0.1 M Na HEPES pH 7.5, 1.8–2.2 M ammonium sulfate, 2% PEG 400; 0.1 M Na HEPES pH 7.5, 1.5–1.8 M ammonium sulfate, 0.2 M NaCl; 0.1 M Na MES pH 6.5, 1.8–2.0 M ammonium sulfate, 5% PEG 400; 0.1 M Na MES pH 6.5, 1.5–1.8 M ammonium sulfate). In one condition, comprised of 0.1 M Na MES pH 6.5, 1.8 M ammonium sulfate and 5% PEG 400, one crystal with an appropriate size for data collection (40 × 110 × 200 µm) was obtained after 4 d (Fig. 1).
Figure 1 Crystals of the haloalkane dehalogenase DppA grown by the hanging-drop vapour-diffusion method. The crystal which was used for data collection (the leftmost crystal) has a thickness of approximately 40 µm. The solid bar represents 100 µm. (more ...)
This crystal of DppA diffracted X-rays to a resolution of 1.95 Å on a rotating-anode X-ray generator. The integration limit was set to an average I/σ(I) of 2. The data statistics are reported in Table 1.
X-ray data-collection statistics for DppA
The crystal of DppA belonged to the orthorhombic space group P21212 (No. 18), with unit-cell parameters a = 47.3, b = 108.5, c = 67.3 Å.
Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient V
M was calculated to be 2.6 Å3 Da−1.
The structure could be solved by molecular replacement using the program Phaser
(McCoy et al.
) with the haloalkane dehalogenase from X. autotrophicus
GJ10 (PDB code 1edb
; Verschueren et al.
), which has a sequence identity of 50%, as a search model. A unique solution was obtained with a monomer in the asymmetric unit with a likelihood score of 483.81 and a Z
score of 21.51. Refinement is currently in progress.