Seed bead kit

Crystallization trials for the Ric-8A:ΔN31Gαi1:Nb8109:Nb8117:Nb8119 complex were conducted by vapor diffusion using commercially available crystallization screening kits. Sitting drops were set on 96-2 well INTELLI-PLATEs (Art Robbins Instruments) using a Gryphon crystallization robot (Art Robbins Instruments) at 3–10 mg/mL protein complex at a 1:1 v/v ratio with precipitation solution. Initial crystallization conditions were identified from hits on the ShotGun screen (MD1-88 Molecular Dimensions). Further optimization was carried out by grid screening variations in sodium malonate (Hampton Research) concentration and pH and by crystal seeding by hanging drop on 24-well VDXm plates (Hampton Research). Crystal seed stocks were prepared with the Seed Bead kit (Hampton Research) in 1.4 M sodium malonate pH 6.9. 0.9 μl of protein stock was added to 0.6 μl of reservoir and 0.3 μl of crystal seed stock and incubated at 12 °C for a minimum of 1–2 weeks. Optimal crystals were obtained from hanging drops containing 3.6 mg/ml Ric-8A:ΔN31Gαi1:3Nb, 1.4 M sodium malonate pH 6.9 at 12 °C.

Crystallization of purified LceB (22–366) was screened for utilizing commercially available screens (NeXtal) and a Crystal Gryphon robot (Art Robbins Instruments). Crystals of LceB grew in gel filtration buffer and 0.1 M Citric Acid pH 4.0, 10% MPD in a 1:1 drop ratio at 15 and 10 mg/mL of protein at 4°C. These conditions were optimized using the sitting drop vapor diffusion method at 4°C (moved to 20°C 5 days later), with crystals generated in varying concentrations of citric acid (0.04 M‐0.15 M) pH 4.0 and MPD (8%–12%) (mother liquor) at a concentration of 10 mg/mL. As the optimized crystals were of insufficient size, they were micro‐seeded using the SeedBead kit (Hampton Research). Crystals were obtained in gel filtration buffer 50 mM Tris 750 mM NaCl pH 7.5 at 20°C from 0.1 M citric acid pH 4.0, 10% MPD at a concentration of 7 mg/mL and used for subsequent data collection and refinement.

The protein was first crystallized in 0.1 M bis-Tris pH 6.5, 22% PEG 3350. Several crystal ‘flower’ aggregates were collected from this condition and were used to generate microseeds using a Hampton Research Seed Bead kit. For room-temperature X-ray crystallography, crystals were grown in 10 µl sitting drops made by mixing the protein sample (5 mg ml⁻¹) and reservoir solution (0.1 M bis-Tris pH 6.5, 18–20% PEG 3350) in a 1:1 ratio with 0.2 µl of microseeds (1:200 dilution). This condition was formulated to produce a pH of 7.0 in the crystallization drop, which was confirmed by direct pH measurement using a microelectrode. Single plate-like crystals grew in several days and continued to grow at 14°C. To obtain fresh crystals with a native (non-oxidized) Cys145, a batch setup was used to grow crystals in 200 µl drops containing protein at 6.6 mg ml⁻¹ in 10% PEG 3350, 0.1 M bis-Tris pH 7.0 with 0.4 µl of microseeds.

Single-chain JAK2/EPOR purified into a final buffer of 10 mM Hepes pH 7.2, 200 mM NaCl and 1 mM TCEP was highly insoluble, with near complete precipitation observed upon concentration. Upon further inspection with phase contrast light microscopy, the precipitation was determined to be crystalline in nature. These microcrystals were pelleted by centrifugation and dissolved by addition of 100 mM Citric acid pH 5.5. Lowering the pH enabled concentration of the protein to 7 mg/mL. After sparse matrix screening, a single initial hit was obtained in 100 mM Tris pH 8.5, 8% PEG8000. Subsequent preparations of JAK2/EPOR were subjected to final SEC in 10 mM Na Citrate pH 5.5, 200 mM NaCl, 1 mM TCEP to reduce spontaneous crystallization during purification. Diffraction quality crystals were obtained by microseeding, using the Seedbead kit (Hampton Research) into 100 mM Tris pH 7.6, 2–4% PEG8000. Crystals were cryoprotected in mother liquor supplemented with 30% ethylene glycol.
For crystallization of the JAK2/LEPR complex, protein was concentrated to 9 mg/mL in final SEC buffer. Diffraction quality JAK2/LEPR crystals were obtained in 0.1 M MES pH 6.5, 0.2 M MgCl₂, 5–10% PEG4000, and 10% ethylene glycol using microseeding and PEG4000 dehydration up to 10% PEG4000. Crystals were cryoprotected in mother liquor with a final concentration of 25% ethylene glycol.

All crystals were grown using the hanging-drop method by mixing 1.5 μL of reservoir solution mixed with 1.5 μL of protein solution at RT. Crystals of native and Se-Met mouse lipin 1 M-Lip^xtal domain were grown in 5–15% PEG 3000, 0.1 M Tris-HCl, pH 7.0 by seeding with microcrystals generated using a Seed Bead Kit (Hampton Research, HR4-781). Crystal form 2 of mouse lipin 1 M-Lip^xtal were grown in 0.2 M zinc acetate, 1 M NaCl, 0.1 M Imidazole, pH 8.0 by seeding with microcrystals. Crystals of mouse lipin 2 M-Lip^xtal were grown in 0.2 M Calcium Acetate, 20% PEG 3350. All crystals were cryoprotected with 25% v/v glycerol and flash frozen in liquid nitrogen prior to data collection. Diffraction data for mouse lipin 1 native M-Lip^xtal domain was collected at 0.979 Å at the APS NE-CAT 24-ID-C beamline (Table 1). Data for the Se-Met and crystal form 2 of the mouse lipin 1 M-Lip^xtal domain were collected at 0.979 Å and 1.28 Å at the NSLS-II FMX 17-ID beamline at Brookhaven National Laboratory (Table 1). Diffraction data for mouse lipin 2 M-Lip^xtal domain was collected at 0.979 Å at the Advanced Photon Source GM/CA ID-23B beamline at Argonne National Laboratory (Table 1).

Compound YS0470 was prepared as a 100 mM solution in 100 mM Tris–HCl pH 7.5, and MgCl₂ was prepared as a 100 mM aqueous solution. These solutions were added to the hFPPS sample to give final concentrations of 1 mM inhibitor, 1.5 mM MgCl₂ and 0.25 mM (10 mg ml⁻¹) protein. Crystals suitable for X-ray diffraction were obtained at 295 K by vapour diffusion in a sitting drop composed of 1 µl inhibitor/MgCl₂/protein mixture, 1 µl crystallization solution (30% PEG 400, 2 M ammonium phosphate, 0.2 M MgCl₂, 0.1 M HEPES pH 7.5) and 0.5 µl seed stock. The seed stock was prepared with a Seed Bead kit (Hampton Research) using a crystal grown in a sitting drop consisting of 1 µl ligand-free protein sample (10 mg ml⁻¹) and 1 µl crystallization solution (2.0 M ammonium phosphate, 0.1 M Tris–HCl pH 8.5).

Recombinant FL_L27D was treated with 1 U µg⁻¹ thrombin (New England Biolabs) overnight to remove the hexahistidine purification tag. Subsequently, thrombin and the cleaved peptide tag were separated from FL_L27D by size-exclusion chromatography. Sitting-drop vapor-diffusion crystallization trials were set up using a Mosquito crystallization robot (SPT Labtech) in nanolitre-scale Swissci 96-well triple-drop plates (Table 2 ▸). The protein plates were incubated at 293 K. Commercially available sitting-drop crystallization screens from Molecular Dimensions and Hampton Research were used to screen for conditions allowing crystal nucleation and growth. Seeding of crystallization conditions was performed using the Seed Bead Kit (Hampton Research) following the contemporary protocol.