Computational protocol: Structural Basis of the Chromodomain of Cbx3 Bound to Methylated Peptides from Histone H1 and G9a

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Protocol publication

[…] CBX 3 (26 mg/ml) and the trimethylated lysine peptide were mixed in the ratio 1∶1.2 and incubated overnight at 4°C and the hanging drop was set up against the well solution of 40% PEG 550MME at 298 K at 1∶1 ratio. Crystals appeared after 4 days and were flash frozen in liquid nitrogen and the data was collected to 2.4 Å and 1.8 Å, respectively.The data was processed with HKL2000 and scaled with Scalepack . Results can be found in the files den_out.pdf and scale_log.pdf attached. The data integrated and scaled in the space group P3221 was converted to mtz using the programs truncate and unique. Molecular replacement was then performed using the program Amore using a model of the structure of Drosophila Polycomb chromodomain (PDB code: 1PDQ). A good solution with four molecules in the asymmetric unit was obtained. The program RESOLVE was used to improve the phases obtained with the refined Amore model and ARP_WARP built 95% of the molecule.CBX3 structure was obtained by molecular replacement and traced by ARP_WARP . Model building, model completion and validation were then performed using the program Coot . A final validation was done using MolProbity . […]

Pipeline specifications

Software tools ARP/wARP, Coot, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens