Computational protocol: Chromatin Insulator Factors Involved in Long-Range DNA Interactions and Their Role in the Folding of the Drosophila Genome

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Protocol publication

[…] Crystallization trials was carried out by the sitting-drop technique using the classic, PEG, PACT and AmSO4 suites (Quiagen, France) and low-profile microplates (Grenier, France) at room temperature. 0.5 µl protein solution was mixed with an equal volume of reservoir solution. Several conditions yielded crystals. Optimizations were done with the hanging-drop vapor diffusion technique. 1 µl protein solution was mixed with 1 µl of reservoir. We obtained well diffracting crystals (2.03 Å) using 0.8 M NaH2PO4, 0.8 M KH2PO4, 0.1M Hepes/pH 7.5. Crystals were soaked in 30% glycerol for cryoprotection and diffraction data were collected under cryogenic conditions on our laboratory anode and at the European Synchrotron Radiation Facility (ESRF, Grenoble). Image data were processed and scaled using the programs MOSFLM (Leslie, 1999) and SCALA of the CCP4 suite . The crystal belonged to space group P3221 with unit cell parameters a = b = 84.98 Å, c = 40.87 Å, α = β = 90° and γ = 120°.The structure of CP190-BTB/POZ was solved by molecular replacement with an in-house dataset at 2.3 Å resolution using the program PHENIX (phenix.autoMR) and a combination of five partial models extracted from the server TOME used to gather potential templates through fold-recognition. Structure refinement and rebuilding were performed with COOT , PHENIX (phenix.refine) and REFMAC (Murshudov et al, 1997) from the CCP4 suite using a dataset recorded at the ESRF at 2.0 Å resolution. Data collection and refinement statistics are summarized in Supplementary . The structure has been deposited with the Protein Data Bank (PDB 4U77). […]

Pipeline specifications

Software tools SHELX, iMosflm, CCP4, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Drosophila melanogaster