Computational protocol: Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies

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Protocol publication

[…] For crystallization, BL21(DE3)/pLysS cells carrying pR1EN were cultured in LB medium at 37°C. When OD600 had reached to 0.6, IPTG was added to the medium at final concentration as 0.75 mM, and cells were further incubated for 6 h at 26°C. Cells were collected by centrifugation and stored at −80°C.Stored cells were thawed and sonicated in lysis buffer [40 mM Tris–Cl (pH 7.5), 0.5 M NaCl, 50 mM imidazole, 0.1% triton X-100 and 0.1 mM PMSF], centrifuged and the supernatants were subjected to Nickel-trapped HiTrap Chelating column (GE healthcare) and eluted by elution buffer [40 mM Tris, 0.5 M NaCl, 0.35 M imidazole, (pH7.5)]. The proteins were precipitated by adding ammonium sulfate and collected by centrifugation, and the pellet was dissolved with 10 ml of digestion buffer [50 mM Tris–Cl (pH 7.5), 0.3 M NaCl, 1 mM DTT, 2 mM CaCl2], followed by Factor Xa (New England Biolab) digestion for overnight at 10°C.The sample solution was loaded on HiTrap SP column (GE healthcare) followed by Superdex 200 (GE healthcare), resulting the highly purified R1Bm EN. The protein was concentrated in sample buffer (40 mM HEPES-Na pH 7.2, 200 mM NaCl, 1 mM DTT, 1 mM EDTA) with 10 kDa Microcon (Millipore) and protein solution was prepared to 6 mg/ml. In that case, the concentration of R1Bm EN was determined by absorption of 280 nm spectra with extinction coefficients of 27 310 M−1 cm−1.Crystals of the R1Bm EN were obtained by hanging drop vapor diffusion method at 283 K. Two microliters of protein solution were mixed with 1.5 μl of reservoir solution containing 2.4 M sodium acetate (pH 6.9), 10 mM ammonium sulfate, and 1–2% Jeffamine M-600 reagent. The hexagonal rod-shaped crystals, whose size of 0.2 × 0.2 × 0.5 mm3, were gown within 2–3 weeks.Prior to the data collection, crystals were transferred into cryoprotectant solutions containing 9% (v/v) ethylene glycol and 9% (v/v) PEG-200 in reservoir solution and flash frozen in cryo nitrogen stream. Dataset was collected at the Photon Factory (Tsukuba, Japan) on beamline BL-6A. Data processing and reduction were carried out with the programs MOSFLM () and SCALA (). Phases were calculated by molecular replacement method with the MolRep () program using TRAS1-EN structure (PDB entry: 1WDU) as a search model. Further model was built with program O () and structure refinement calculations including simulated annealing and B-factor refinement were done with CNS (). Summary of the crystallographic statistics are shown in . […]

Pipeline specifications

Software tools CCP4, Molrep, CNS
Application Protein structure analysis
Organisms Bombyx mori