Computational protocol: Modification dependent restriction endonuclease, MspJI, flips 5 methylcytosine out of the DNA helix

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Protocol publication

[…] MspJI wild type (WT) was expressed and purified as previously described (,). For co-crystallization, final concentrations utilized were ∼12 mg ml−1 MspJI (0.24 mM monomer or 0.06 mM tetramer) in 20 mM Tris-HC1 (pH 8.0), 150 mM NaCl, 10% glycerol (v/v), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT) and 0.13 mM 27-bp oligo, in the presence of 4 mM CaCl2 (to avoid cleavage of the oligo). Crystals were grown at 16°C by the hanging-drop vapor-diffusion method using equal amounts of protein/DNA mixture and well solutions with 10–15% polyethylene glycol MME 2000, 12.5–15% TacsimateTM (Hampton Research), pH 6.4–6.6. The Tacsimate solution (100% at pH 7.0) was adjusted with HCl to lower the pH to 6.4–6.6. Crystals large enough to acquire with a nylon loop (Hampton) were quickly transferred to the well solution containing 20% ethylene glycol before being flash-frozen directly in liquid nitrogen, stored and later used in X-ray diffraction experiments.The data set was processed using the program HKL2000 (). During initial processing, data first appeared to be P6122, but were determined to be P61 by merohedral twinning and Matthews probability analysis. Molecular replacement with the MspJI tetramer (PDB 4F0Q) () as the initial search model, map production and model refinement were conducted using the PHENIX software suite (). As the twin fraction refinement revealed the data to exhibit near perfect merohedral twinning, we proceeded with map examination and refinement carefully using restraints for secondary structure and NCS (non-crystallographic symmetry) afforded in PHENIX. Restraints were relaxed in later rounds of refinement. Only group thermal B-factor refinement was utilized until the last rounds of refinement, where individual thermal B-factors refinement and TLS (translation/libration/screw) parameters were applied. Maps and models were visualized with COOT () as well as conducting manual model manipulation during refinement rounds. […]

Pipeline specifications

Software tools PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Chemicals Cytosine, Nucleotides, 5-Methylcytosine