Index ht

The initial crystallization screening of RdFucI (30 mg/ml) was performed with the Index HT, Salt RX HT, and Crystal Screen HT commercially available kits (Hampton Research, Aliso Viejo, CA) using the sitting-drop vapor-diffusion method at 20 °C. Microcrystals were obtained by precipitation in a solution containing 0.1 M HEPES, pH 7.5, and 20% (w/v) polyethylene glycol 10,000. Suitable crystals for X-ray diffraction were obtained using the diluted RdFucI (15 mg/ml) solution with the crystallization solution using the hanging-drop vapor-diffusion method at 20 °C.

The DNA fragment coding for the CgT protein (residue 287–582) was cloned into pET-sumo vector with a primer set of CgT-sumo-BamH1-F and CgT-sumo-Xho1-R (Table 2), and expressed in E. coli BL21(DE3), purified as previously described (25 (link), 47 (link)), and stored at 4 °C in a buffer that contained 20 mm Tris-HCl, pH 8, 100 mm NaCl, and 0.2 mm Tris(2-carboxyethyl)phosphine. Crystallization screens were conducted using the sitting drop vapor diffusion method with a Phoenix crystallization robot on a 96-well Intelli-plate (Art Robbins Instrument). Designed crystallizations were set up manually using the vapor diffusion hanging drop method with 24-well plates. The drops were set up at a 1:1 ratio of protein to mother liquor and incubated at 20 °C. Crystals grown in a well with 0.1 m sodium citrate tribasic dehydrate, pH 5.5, and 22% PEG1000 of IndexHT (Hampton Research) were used to determine the CgT structure.

Screening for crystallization conditions was performed by sitting-drop vapor diffusion in two 96-well plates using the Index HT (Hampton Research) and JCSG-plus (Molecular Dimensions) screens at 293 K. Various conditions led to crystallization, with most containing MgCl₂·6H₂O as the precipitant and PEG as a crowding agent. Optimization of the crystallization conditions of 1-Cys SiPrx was performed by the hanging-drop vapor-diffusion method, in which 2 µl protein solution (7.5 mg ml⁻¹ in 10 mM Tris–HCl pH 7.5) was mixed with 2 µl reservoir solution and equilibrated against 200 µl of the same reservoir solution, followed by incubation at 283 K. The optimal reservoir solution was found to be 10 mM Tris–HCl pH 7.5, 15%(w/v) PEG 1500, 0.1 M MgCl₂. Using this condition, diffracting crystals were obtained after a two-week incubation.

Initial crystallization conditions were identified from screening with commercial kits: JCSG+ (Qiagen), PACT (Qiagen), Crystal Screen (Hampton Research) and Index HT (Hampton Research) using an Oryx 8 robot (Douglas Instruments) and a protein concentration of 100 mg ml^-1. The high solubility of the protein is likely due to the glycosylation of the protein. Crystals formed under several conditions, but most of them diffracted poorly. Crystals obtained from condition C2 (30% PEG400, 0.2M MgCl₂, 0.2 M HEPES pH 7.5) in the JSCG⁺⁺² screen from Jena Bioscience were further optimized in a manual setup using grid screens (pH, salt and PEG 400), additives and variation of drop ratios. All crystallization experiments were performed at room temperature, and both sitting and hanging drop vapor-diffusion setups were tested. Replacement of MgCl₂ with CaCl₂ improved the quality of the crystals. The crystal used for data collection was grown in a hanging drop setup with 3 μl protein (66 mg ml^-1) and 2 μl reservoir solution equilibrated over a 500 μl reservoir (0.1 M HEPES pH 7.5, 34% PEG 400, 0.22 M CaCl₂, 0.05 M glycine). Attempts were made to soak the substrates 2,6-dimethoxy phenol (2,6-DMP) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) into the crystals by adding solid substrate to the drop as described by Kallio et al. [8 (link)].

Protein crystallization was carried out by the vapor-diffusion method at 20°C, mixing 1 μL protein with 1 μL well solution. The purified protein was subjected to a high-throughput crystallization screening using a Mosquito crystallization robot (TTP Labtech, UK) and commercially available 96-well kits: Xtal Quest (BioXtal), Wizard (Rigaku), Crystal Screen (Hampton Research) and Index HT (Hampton Research).
Crystals appeared after several days. mtCfaA -CfaE was crystallized using a well solution containing 2% PEG8000, 0.1 M imidazole malate, 1.04 M Lithium Sulfate and 0.001 M GSSG-GSH. All crystals were flash-cooled in liquid nitrogen in the presence of 20–30% glycerol. Diffraction data sets were collected at the SER-CAT ID22 beamline at the Advanced Photon Source (APS), Argonne National Laboratory (ANL) with a MAR300 CCD detector.

Crystallization screenings were performed using high-throughput techniques in a NanoDrop robot and Innovadyne SD-2 microplates (Innovadyne Technologies, Inc.) with screening PACT Suite and JCSG Suite (Qiagen), JBScreen Classic 1 to 4 and 6 (Jena Bioscience), and Crystal Screen, Crystal Screen 2, and Index HT (Hampton Research). The conditions that produced crystals were optimized with a sitting-drop vapor-diffusion method at 291 K by mixing 1 μl of protein solution and 1 μl of precipitant solution, equilibrated against 150 μl of precipitant solution in the reservoir chamber. The best crystals were obtained in a crystallization condition containing 0.15 M sodium acetate, 0.1 M Bis-Tris propane (pH 6.5), and 16% (wt/vol) PEG3350 (Fig. S1B). Protein concentration was assayed at the concentration of 8 mg/ml.