All EsxB proteins were screened for crystallization conditions with the help of a Mosquito nanoliter liquid handler (TTP LabTech) using the sitting drop vapor diffusion technique in 96-well CrystalQuick plates (Greiner). For each condition, 0.4 µL of protein and 0.4 µL of crystallization formulation were mixed; the mixture was equilibrated against 140 µl of the crystallization solution in each reservoir well. The crystallization screens used were MCSG-1–4 at 4 and 24°C. Diffraction quality crystals appeared under conditions including: (1) SeMet-labeled WT-EsxB-His6: 0.1 M Na2HPO4:citric acid, 2.0 M ammonium sulfate, pH 4.2; (2) WT-EsxB-His6: 0.1 M sodium acetate, 2.0 M ammonium sulfate, pH 4.5; (3) SeMet-labeled WT-EsxB: 0.1 M phosphate citrate, 40% (v/v) PEG 300, pH 4.2; (4) WT-EsxB: 0.2 M NaCl, 0.1 M Tris:HCl, 30% (w/v) PEG 3000, pH 7.0; (5) Y65F: 0.1 M Bis-Tris propane, 2 M di-ammonium hydrogen citrate, pH 7.0; (6) P67A: 0.1 M sodium citrate:citrate acid, 40% (v/v) PEG 600, pH 5.5; (7) G53A: 0.1 M sodium acetate:HCl, 25% (w/v) PEG 3350, pH 4.5; (8) E54Q: 0.17 M ammonium acetate, 0.085 M sodium acetate:HCl, 25.5% (w/v) PEG 4000, 15% glycerol, pH 4.6. Prior to data collection, the crystals were treated with cryoprotectants and cryocooled directly in liquid nitrogen.
Mosquito nanoliter liquid handler
Manufactured by SPT Labtech
The Mosquito nanoliter liquid handler is a precise and automated liquid handling instrument designed for low-volume liquid transfer. It is capable of accurately and consistently dispensing nanoliter-scale volumes of liquids, making it suitable for various applications that require precise and reproducible liquid handling.
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5 protocols using mosquito nanoliter liquid handler
1
EsxB Protein Crystallization Conditions
2
Optimized Crystallization of CthHsp104 Variant
3
Optimizing Protein Crystallization Conditions
4
Crystallization of MtbTOP1-704t-ssDNA Complex
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MtbTOP1-704t was concentrated to ∼1.1 mM (∼85 mg/ml). The protein, MTS2-12 oligonucleotide (5′-CTTCCGCTTGAC-3′, custom synthesized by Sigma-Aldrich, ∼2 mM) and MgCl2 (from 50 mM stock solution) were mixed at a molar ratio of 0.55:1.25. The mixture was then incubated on ice for ∼2 h. With a Mosquito nanoliter liquid handler (TTP LabTech), crystallization condition screening was set up using the sitting drop vapor diffusion method with four 96-condition screens: Index, PEG/ION and SaltRx from Hampton Research; and NeXtal PEGs II from Molecular Dimensions, at 16°C. Each crystallization droplet containing 0.4 μl of protein–DNA mixture, and 0.4 μl of precipitating agent was equilibrated against a reservoir containing 140 μl of precipitating solution. About 10 days after the setting, typical needle-like crystals appeared in crystallization drops under several conditions. The structures of these crystals were determined to be the same as the MtbTOP1-704t–ssDNA complex as reported previously (PDB codes: 6CQI and 6CQ2) (26 (link)). About 8 months later, a single rectangular-shaped crystal appeared under a new condition comprising 0.1 M sodium acetate trihydrate, pH 7.0 and 12% (w/v) polyethylene glycol (PEG) 3350. This crystal was harvested and treated with a cryoprotectant solution (25% glycerol in its mother liquor) and then flash-frozen in liquid nitrogen before data collection.
5
Structural Characterization of MsmTOP1-839t Protein
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The purified MsmTOP1-839t protein (92.3 kDa) was concentrated to about 38.8 mg/ml (∼0.43 mM) for crystallization. For the co-crystallization trials with ssDNA, the protein was first mixed with ssDNA MTS2-25 (Table 1 ) in a 1:2 molar ratio and then incubated on ice for 2 h before crystallization set-up. Screening for crystallization conditions was set up with a Mosquito nanoliter liquid handler (TTP LabTech) using the sitting drop vapor diffusion technique in 96-well CrystalQuick plates (Greiner). For each condition, 0.2 μl of MsmTOP1-839t/MTS2-25 and 0.2 μl of crystallization formulation were mixed; the mixture was equilibrated against 140 μl of the crystallization solution in each reservoir well. The crystallization screens used were MCSG-1–4 at 16°C. Crystals appeared under several conditions including crystals under the condition of 0.1 M Tris–HCl pH 8.5 and 1.5 M lithium sulfate. One of the crystals from this condition diffracted to the highest resolution limit (3.1 Å) as described below. For the preparation of the crystals for X-ray diffraction experiments, they were harvested and transferred to cryoprotectant solution that contains 25% glycerol in addition to crystallization buffer for a few minutes and then cryocooled directly in liquid nitrogen.
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