Computational protocol: Structural basis for the recognition and cleavage of histone H3 by cathepsin L

Similar protocols

Protocol publication

[…] Single chain, mature C25A (mC25A) was incubated with a 5 molar excess of each peptide for 30 min at 4 °C. Crystallization conditions were identified by means of sparse matrix screening using the sitting drop vapour diffusion method. The concentration of mC25A was 7 mg ml−1. Diffraction quality crystals were obtained by mixing equal ratios of protein and reservoir solutions. The final crystallization condition for the apo-mC25A crystals was 0.2 M ammonium sulphate, 0.1 M sodium acetate trihydrate, pH 4.6, 30% polyethylene glycol 2000 monomethylether. Crystals for the mC25A-H319−33 complex were obtained in 10% isopropanol, 0.1 M sodium acetate trihydrate, pH 4.0, 22% polyethylene glycol 6000. All crystals were grown at 18 °C and required approximately 4 weeks to reach optimal dimensions.All crystals were soaked in 25% glycerol, added to the final crystallization solution and frozen in liquid nitrogen. Synchrotron diffraction data for the apo-mC25A and mC25A-H319−33 crystals were collected at beamline 23-ID-B, Advanced Photon Source, Argonne National Laboratories. All data were indexed and scaled in HKL2000. Three-dimensional structures were determined by means of molecular replacement with the wild-type cathepsin L structure derived from the PDB 3bc3 deposition using PHASER. Structure building and refinement were carried out with Coot and REFMAC5, including TLS (translation, liberation and screw-rotation) refinement with appropriate non-crystallographic restraints. The data collection and structure refinement statistics are presented in . Data for the mC25A-H319−33 complex were severely anisotropic. Data were collected with 95% completeness in the 2.82–2.69 Å shell and with 78.9% completeness in the 2.69–2.59 Å shell. Owing to the low Rmerge and high I/sig(I) ratio, data binned in the 2.59–2.5 Å shell were included in the refinement. All structural figures were generated with PyMOL. […]

Pipeline specifications

Software tools Coot, REFMAC5, PyMOL
Application Protein structure analysis
Organisms Mus musculus, Homo sapiens