Peg ion screen 1

Crystal pre-screening of BLEG-1 was carried out using the sitting drop vapor diffusion method at 4 °C with the following commercial screening kits: Crystal Screen 1™ and Crystal Screen 2™ (Hampton Research, Aliso Viejo, CA, USA), Index Screen™ (Hampton Research, Aliso Viejo, CA, USA), PEG/Ion Screen 1™ and PEG/Ion Screen 2^TM (Hampton Research, Aliso Viejo, CA, USA), and JCSG plus™ (Molecular Dimensions, Sheffield, UK). Purified BLEG-1 at concentrations of 3 and 17 mg/mL, respectively, was used at 1:1 and 2:1 protein:formulation ratios to identify the suitable protein concentration and formulation for BLEG-1 crystal growth. Further optimization was performed by varying PEG3350 concentrations: 10%, 20%, and 30% (w/v); and NaI concentrations: 0.1, 0.2, 0.3, 0.4, and 0.5 M using the formulation that formed protein crystals. Incubation temperature for crystal growth was also screened at 4 °C and 20 °C, respectively. Single protein crystal obtained at the optimum conditions was selected and diffracted with an in-house X-ray diffractometer (Rigaku Micromax-007 generator) equipped with a CCD MSC PAD200sk detector at Malaysian Genome Institute, Selangor, Malaysia, at 100 K and wavelength of 1.54 Å. The diffraction data was processed and scaled with Denzo and SCALEPACK in the HKL 3000R software (version v714n, HKL Research Inc., Charlottesville, VA, USA) [48 (link)], respectively.