Computational protocol: Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation

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Protocol publication

[…] For co-crystallization with the 28-bp DNA, 0.35 mM of TDG wild-type protein was mixed with 0.2 mM of annealed oligonucleotide (synthesized by the New England Biolabs, Inc.): 5′-CAG CTC TGT ACG TGA GCG ATG GAC AGC T-3′ and 5′-AGC TGT CCA TCG CTC AXG TAC AGA GCT G-3′ where X is 5hmU. The 5-hydroxymethyl-deoxyU phosphoramidites used for DNA synthesis were purchased from Glen Research. Crystals appeared within 24 h under the conditions of 30% polyethylene glycol (PEG) 4000, 0.2 M ammonium acetate, 0.1 M sodium acetate, pH 4.6. Crystals were cryoprotected by soaking in mother liquor supplemented with 20% ethylene glycol. X-ray diffraction datasets were collected at the SER-CAT beamline (22ID-D) at the Advanced Photon Source, Argonne National Laboratory and processed using HKL2000 (). The structures were solved by molecular replacement by PHENIX () using TDG and the abasic DNA complex structure [PDB 2RBA ()] as the search model. Electron density for DNA was easily interpretable, and its model was built using the programs O and Coot (). PHENIX refinement scripts were used for refinement, and the statistics shown in Supplementary Table 1 were calculated for the entire resolution range. The Rfree and Rwork values were calculated for 5% (randomly selected) and 95%, respectively, of observed reflections. […]

Pipeline specifications

Software tools PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Escherichia coli
Chemicals Cytosine, Guanine, Hydrogen, Thymine, Uracil, 5-Methylcytosine