Crystal screen 1 2

Crystallization was performed using the microbatch crystallization method under a thin layer of Al’s oil, using the commercially available screening kits Wizard I, II (Emerald BioSystems, Washington, USA) and Crystal Screen I, II (Hampton Research, CA, USA) at 295 K. Diffraction-quality crystals were obtained from the condition Wizard I-34 (1.0 M ammonium phosphate dibasic, 0.1 M imidazole pH 8.0) and Crystal Screen I-42 (50 mM potassium phosphate monobasic, 20% w/v polyethylene glycol 8000). Crystals were immediately frozen in a cold nitrogen stream for MAD data collection. For X-ray data collection, the CcEstA crystal was transferred in the cryosolvent containing the same reservoir and 20% glycerol before being flash-frozen in a cold nitrogen stream. X-ray diffraction data were collected at 100 K using an ADSC Quantum 315 CCD on beamline PAL 4 A at the Pohang Accelerator Laboratory (PAL), Korea. The data were indexed, integrated and scaled using HKL-2000. The data collection and processing statistics are summarized in Table 1.

The purified pAnpl-UAA*01 complex was ultimately concentrated to 10 mg/ml. After mixing with reservoir buffer at a 1:1 ratio, the purified protein was crystallized using the sitting-drop vapor diffusion technique at 277 K. Index, Crystal Screen I/II, and Crystal Screen Cryo I/II kits (Hampton Research, Riverside, CA) were used to screen for optimal crystal growth conditions. After several days, crystals (pAnpl-UAA*01–MVM9 and pAnpl-UAA*01–RLI9) were observed with solutions NO.7 from the Crystal Screen Cryo II kit (8% polyethylene glycol 1000, 8% [wt/vol] polyethylene glycol 8000, and 20% [vol/vol] glycerol) and NO.43 from the Crystal Screen Cryo I kit (24% [wt/vol] polyethylene glycol 1500 and 20% [vol/vol] glycerol), respectively. Diffraction data for pAnpl-UAA*01 crystals were collected to resolutions of 1.71 Å (pAnpl-UAA*01–MVM9) and 2.06 Å (pAnpl-UAA*01–RLI9) at the Shanghai Synchrotron Radiation Facility (SSRF) using beamline BL17U at a wavelength of 1.5418 Å (Shanghai, China) (64 (link)).The crystals were first soaked in reservoir solution containing 25% glycerol as a cryoprotectant and were then flash-cooled in a stream of gaseous nitrogen at 100 K (65 (link)). The collected intensities were indexed, integrated, corrected for absorption, scaled, and merged using the HKL2000 package (66 (link)).

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.