Phoenix crystallization robot

BpumGH48 crystals in complex with cellobiose (BpumGH48-C2) and cellobiose/cellohexaose (BpumGH48-C2C6) were initially obtained with sitting drop vapor diffusion using a 96-well plate with Grid Screen Salt HT from Hampton Research (Aliso Viejo, CA). 50 µL of well solution was added to the reservoir and drops were made with 0.2 µL of well solution and 0.2 µL of protein solution using a Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, CA). The crystals were grown at 20 °C using screens containing 1–3 M malonate with pH 5–7 and 20 mM cellobiose. The protein solutions contained 15 mg/mL of protein in 20 mM acetate buffer pH 5, with 100 mM NaCl and 10 mM CaCl₂. Before freezing crystals were briefly soaked in a drop containing excess amounts of cellohexaose, 10% (v/v) glycerol and 10% (v/v) ethylene glycol.

Initial crystallization screens were performed using a Phoenix crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits (Hampton Research, Qiagen, or Emerald BioSystems), followed by extensive manual optimization. The best single crystals were grown at 18 °C by the hanging-drop vapor-diffusion method in a 1:1 (v/v) ratio of protein and reservoir, as follows. (1) 4H11-scFv was crystallized with a reservoir solution composed of 0.1 M sodium citrate tribasic dihydrate (pH 5.0) and 20% polyethylene glycol (PEG) 4 K. Micro-seeding was necessary to obtain single crystals. (2) 4H11-scFv-MUC16-target complex was crystallized using a reservoir of 0.1 M sodium citrate tribasic dihydrate (pH 5.0), 10 mM barium chloride dihydrate, and 27% methoxypolyethylene glycol 5000 (PEG MME 5 K).

Crystallization experiments were performed with a stock solution of purified MG Orn at 3.6 mg·mL⁻¹ in 20 mm HEPES pH 7.5 (at 25 °C), 150 mm NaCl, 1 mm MnCl₂. Initial crystallization conditions were screened using the vapor diffusion method set up by a Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, CA, USA). The plates were set up with 60 μL reservoir solution and sitting drops with equal amounts of reservoir solution mixed with protein stock solution in a total drop volume of 0.5 μL. The screens were incubated at 4 °C. Diffraction‐quality crystals were found after 2 weeks at a condition containing 0.1 m HEPES pH 7 (at 25 °C) and 5% PEG 8000. Crystals were harvested, transferred through a cryoprotectant solution consisting of the reservoir solution with 30% (v/v) glycerol added, and flash‐cooled in liquid N₂. X‐ray diffraction data were collected at BL14.1 operated by the Helmholtz‐Zentrum Berlin (HZB) at the BESSY II electron‐storage ring (Berlin‐Adlershof, Germany; Ref.23). The data were indexed and integrated by xds/xscale24, before being merged and scaled by programs in the ccp4 program suite 25. Data collection and processing statistics are presented in Table 1.

PDC_2.03 crystals were initially obtained with sitting drop vapor diffusion using a 96-well plate with Grid Screen Salt HT from Hampton Research (Aliso Viejo, CA). Fifty microliter of well solution was added to the reservoir, and drops were made with 0.2 µL of well solution and 0.2 µL of protein solution using a Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, CA). The crystals were grown in 0.1 M MES monohydrate pH 6.0 and 2.4 M ammonium sulfate at 20 °C. The protein solutions contained 6 mg/mL of protein in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 0.5 mM DTT, and 0.1 mM TPP.

Crystallization experiments were performed with a stock solution of purified Asn3‐ISP at 30 mg/mL in 50 mM TrisHCl pH 7.5 (at RT), 50 mM NaCl. Initial crystallization conditions were screened using the vapor diffusion sitting drop method set up by a Phoenix crystallization robot (Art Robbins Instruments). The plates were set up with 60 µl reservoirs solutions and sitting drops with equal amounts of reservoir solution mixed with protein stock solution in a total drop volume of 1 µl. The screens were incubated at 20 °C. Diffraction‐quality crystals were obtained from 6 conditions, as outlined in Supporting Information Table S3.

Crystals of the deglycosylated PfCel7A were initially detected with sitting-drop vapor diffusion crystallization using a 96-well plate, Grid Screen Salt HT from Hampton Research (Aliso Viejo, California, USA) and setup by a Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, California, USA). For the final data collection crystals were grown using a sitting drop setup with 50 µL of well solution in the reservoirs and 0.5 µL of well solution and 1.5 µL of protein solution in the drop. The crystals grew at 20 °C in 0.27 M sodium phosphate monobasic monohydrate and 1.53 M potassium phosphate dibasic pH 4.0 as the well solution. The protein solution contained 15.5 mg/mL of protein in 20 mM acetate pH 5.0, 100 mM NaCl, 50 mM cellobiose, and 5 mM cellohexaose.