Computational protocol: Crystal structure of trioxacarcin A covalently bound to DNA

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[…] Trioxacarcin A was obtained from the marine Streptomyces sp. isolate B8652 by fermentation (). Oligonucleotides were purchased already purified by HPLC from GmbH and used without further purification. Crystals were grown at 40°C in hanging drops by vapor diffusion; we found by experiment that the higher than usual temperature produced better quality single crystals. The solution in the reservoir contained 1.55 M tri-ammonium citrate (pH 7.0) and 30% v/v DMSO. The DNA–drug solution contained 2.5 mM DNA (single-strand concentration), 2.8 mM trioxacarcin A and 25% v/v methanol. The DNA–drug solution was prepared by mixing trioxacarcin A stock solution (containing 5.0 mM trioxacarcin A and 50% v/v methanol) with a 5.6 mM DNA solution in a 1:1 ratio at room temperature and incubation for 3 days at 4°C. The hanging drops prepared from 1 μl DNA-drug solution and 2 μl reservoir solution were equilibrated against 500 μl reservoir solution. Yellow tetragonal crystals grew within 24 h to a size of 0.2 × 0.1 × 0.05 mm. For data collection at 100 K in a nitrogen gas stream, the crystals were transferred to a cryosolution containing 1.55 M tri-ammonium citrate (pH 7.0), 30% v/v DMSO and 15% v/v glycerol. Three different oligonucleotides were used to obtain crystals as described above: native d(AACCGGTT) and brominated d(AACCGG5BrUT) and d(AACCGGT5BrU). In the brominated oligonucleotides thymidine is replaced by 5-bromodeoxyuridine.The DNA–drug complex crystallized in space group P4122 with unit-cell a = b = 37.60 Å and c = 91.62 Å. Data were collected at beamline 14.2 at BESSY, Berlin with a MAR-165 CCD detector. The crystals containing brominated oligonucleotides were used for two Br-MAD experiments. From both crystals, peak and inflection datasets were collected, 180 frames for each dataset with a ϕ-rotation of 1° per frame (see for crystallographic details). In order to avoid radiation damage, an 0.53 mm aluminium filter was used to decrease the intensity of the direct beam. In addition, a native dataset was collected to 1.78 Å resolution from a crystal containing the native oligonucleotide. The datasets were integrated with HKL2000 () and the space group determined by XPREP (Bruker AXS, Madison WI, USA). SHELXD () was used for substructure solution by searching for two bromine atoms with a resolution cutoff at 2.6 Å. Substructure solution succeeded only with the d(AACCGG5BrUT)-derivative, probably because the crystals diffracted better. SHELXE () was employed for phasing and density modification. In the experimental map, the position of thymine could be deduced from the bromine positions from the substructure solution and the anomalous maps calculated using SHELXE. It was also possible to recognize the drug in the experimental map (A). The asymmetric unit consists of one complete duplex with two intercalated trioxacarcins. The graphics program COOT () was used for model building. The structure was refined isotropically with TLS constraints against the native dataset with REFMAC () to Rwork = 22.0% and Rfree = 26.5% (B). Helical parameters were calculated with 3DNA (). were generated using PYMOL (DeLano Scientific LLC, South San Francisco CA, USA). Figure 3. […]

Pipeline specifications

Software tools SHELX, Coot, PyMOL
Application Protein structure analysis
Chemicals Guanine