Pegrx

In brief, 0.6 μl of AtBioZ protein (~15 mg/ml) mixed with 0.6 μl of the screening buffer was subjected to the routine screen of crystallization using the hanging drop vapor diffusion method. Five different screening kits were used: (i) Crystal screen I & II (Hampton Research), (ii) Index comprising 96 reagents (Hampton Research); (iii) PEG/ion, a kit of high purity Polyethylene glycol 3350 in combinations with 48 unique salts (Hampton Research); (iv) PEGRx, a polymer- and pH-based crystallization screen (Hampton Research); and (v) WIZAED screen (Rigaku). As a result, crystals with good diffraction were grown in 0.2 M sodium nitrate and 20% (v/v) PEG 3350 at 16 °C on the 3rd day post-screen. The crystals were harvested and flash frozen in liquid nitrogen with 20% glycerol as a cryoprotectant. Collection of X-ray diffraction data were performed at BL17U1 beamline of Shanghai Synchrotron Radiation Facility (SSRF). Diffraction images were calculated by HKL-2000 program^{65 (link)}. The initial model was solved by molecular replacement (MR) using the structure of Staphylococcus aureus FabH (PDB: 1ZOW) as the searching model^{66 (link),67 (link)}. Model building and crystallographic refinement were conducted with COOT and PHENIX^{68 (link),69 (link)}. The final structure of AtBioZ was solved at 1.99 Å (Table 1) and deposited into the Protein Data Bank (PDB) with the accession entry: 6KUE.

Initial sparse-matrix crystallization screening of wild-type J30 was conducted using the following screens: Berkeley (from Lawrence Berkeley National Laboratory; Pereira et al., 2017 ▸ ), Crystal Screen, Index, Natrix, PEG/Ion, PEGRx, SaltRx (all from Hampton Research) and MCSG-1 (from Microlytic) (Jancarik & Kim, 1991 ▸ ). They were set up using a Phoenix Robot (Art Robbins Instruments). At a concentration of 12 mg ml⁻¹ purified enzyme, the protein was crystallized in 0.1 M trisodium citrate dihydrate pH 5, 15%(w/v) 2-propanol, 10%(w/v) PEG 10 000. Suitably sized crystals were obtained after 3 d of growth at room temperature using the sitting-drop vapor-diffusion method. J30 CCH crystals were prepared in the same manner. All drops consisted of 1 µl protein solution and 0.5 µl reservoir solution.

The protein was crystallized at 15 mg ml⁻¹ as described above. Commercially available crystallization screens (PEG Ion, SaltRX, PEG-Rx and Index (Hampton Research)) and custom screens (PEG-custom and Bis-Tris magnesium formate) were employed. Thin plate crystals appeared in 3 days under many conditions, with the largest (100 × 100 × 10 µm) in 200 mM L-proline, 100 mM HEPES, pH 7.4, 10% w/v PEG 3350 or in 50 mM MgCl₂, 100 mM HEPES, pH 7.3, 30% v/v PEG 550 MME. The crystals were mounted, frozen and diffracted as described above. A single thin plate crystal diffracted to 3.2 Å resolution (I/σ ≥ 2.0) and was initially indexed into a primitive orthorhombic unit cell with dimensions a = 45.9 Å,b = 63.6 Å, c = 165.6 Å, α = β = γ = 90°. The top indexing solution included only 38% of the non-ice reflections. The P2₁2₁2₁ space group readily gave a clear molecular replacement solution but multiple attempts at subsequent extensive refinement were unable to produce R_work/R_free values below 0.27/0.31. The data were rescaled into space group P12₁1 with dimensions a = 46.0 Å,b = 63.6 Å, c = 166.0 Å, α = 90°, β = 90.162°, γ = 90°. This indexing solution also included 38% of the non-ice reflections. Molecular replacement again readily identified a solution. Subsequent refinement using space group P12₁1 resulted in much lower R_work/R_free values (0.23/0.24).

Initial crystallization screening was performed using Crystal Screen, Crystal Screen 2, Index, PEGRx, PEG/Ion (Hampton Research), The PACT Suite (Qiagen) and Wizard I and II (Emerald Bio) by the sitting-drop vapour-diffusion method in 96-well plates. Drops were comprised of 0.1 µl sample and an equal volume of reservoir solution and were equilibrated against 70 µl reservoir solution at 20°C. Subsequent optimization of the initial hit conditions was performed using hanging-drop vapour diffusion at 20°C by changing the pH value of the reservoir solution and the concentrations of the buffer and precipitant, and by using various additives. The hanging drops were set up by mixing 1.5 µl sample solution with 1.5 µl reservoir solution and were equilibrated against 500 µl reservoir solution in 24-well plates. The initial screening yielded crystals in two conditions: (i) 0.1 M bicine pH 8.5, 15%(w/v) PEG 1500 (PEGRx condition No. 21) and (ii) 0.1 M imidazole pH 8.0, 10%(w/v) PEG 8000 (Wizard II condition No. 34). The small and fragile crystals obtained from the former condition could not be improved, while single crystals could be obtained by optimizing the latter condition. Finally, well diffracting crystals (0.2 × 0.2 × 0.2 mm) were produced with reservoir solution consisting of 0.2 M imidazole pH 8.0, 9%(w/v) PEG 8000.