Computational protocol: Structural and Biochemical Bases for the Inhibition of Autophagy and Apoptosis by Viral BCL-2 of Murine γ-Herpesvirus 68

Similar protocols

Protocol publication

[…] The DNA fragments coding for M11 (residues 1–137) and mouse Beclin1 (residues 101–150) were cloned into pET30a (Novagen) and pPROEX HTa (Invitrogen), respectively. From these vectors, a two-promoter vector was constructed for coexpression of the two proteins. The protein complex was produced in E. coli BL21(DE3) strain (Novagen) at 21 °C overnight and purified using a Ni-NTA column (QIAGEN), a Hitrap Q anion exchange column (Amersham Pharmacia) and a Mono Q anion exchange column (Amersham Pharmacia), equilibrated with 20mM Tris-HCl (pH 8.0), 220mM NaCl and 1mM dithiothreitol. Crystals of the complex were obtained by the hanging-drop vapor diffusion method at 24 °C by mixing and equilibrating 1 μl of each of the protein solution (10 mg/ml) and a precipitant solution containing 25% (w/v) polyethylene glycol 3350, 0.2 M magnesium chloride, and 0.1 M imidazole (pH 7.0). Before data collection, the crystals were immersed briefly in a cryoprotectant solution, which was the reservoir solution plus 10% glycerol. A diffraction data set at 2.3 Å resolution was collected on the beamline 4A at the Pohang Accelerator Laboratory, Korea, and processed using the programs DENZO and SCALEPACK []. The structure was determined by the molecular replacement method with the CCP4 version of MolRep [] using the structure of M11 [] as a search model. Subsequently, model building and refinement were carried out using the programs O [] and CNS []. The final model does not include residues 1–4 and 136–137 of M11, and residues 101–105 and 125–150 of Beclin1, whose electron densities were not observed or were very weak. […]

Pipeline specifications

Software tools CCP4, Molrep, CNS
Application Protein structure analysis
Organisms Mus musculus
Diseases Lymphoma