Computational protocol: Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil-containing DNA

Similar protocols

Protocol publication

[…] Crystallization conditions are summarized in . For the free-liganded protein and the DNA–protein complexes (140 µM protein and 700 µM 12-mer DNA), conditions were screened using the Qiagen kits Crystals and then manually optimized at 18°C in hanging drop by mixing equal volumes of the protein or protein–DNA solution with precipitant solution. Crystals were transferred to a cryoprotectant solution (paraffin oil or 20% PEG 400) and flash frozen in liquid nitrogen. Diffraction data were collected at 100K on the PROXIMA I beamline at SOLEIL synchrotron (Saint-Aubin, France) and intensities were integrated with the program XDS () for all crystals except that of AP•G complex which was collected on ID29 beamline at ESRF (Grenoble). Data collection and processing statistics are given in . Structure determination of all crystals was performed by molecular replacement with PHASER () using first the coordinates of the free-liganded structure (PDB code 3IHO) for our free-liganded structure at a better resolution and next our model for the DNA–protein structures. Refinement was performed using BUSTER () and electron density maps were evaluated using COOT (). The refined models include 138 residues (from 437 to 575). Refinement details of the six structures are shown in . Molecular graphics images were generated using PYMOL (http://www.pymol.org). […]

Pipeline specifications

Software tools XDS, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Thymine, Uracil, 5-Methylcytosine