In an earlier study [Smith, Davies, Dodson & Moore (1995). Biochemistry, 34, 415-425] the crystal structure of the d(TGATCA)-nogalamycin complex was determined to 1.8 A and refined with PROLSQ to R = 19.5% against 4767 reflections with F> 1sigma(F). A low-temperature crystallographic study on this complex has now been performed. Native data collection at liquid-nitrogen temperature (120 K) improved the resolution to 1.4 A. The structure has now been refined against these new diffraction data in the resolution range 8-1.4 A using NUCLSQ, PROLSQ, SHELXL93 and X-PLOR, in order to determine to what extent the resulting DNA conformation and associated solvent structure would differ and to examine the suitability of these programs for the refinement of oligonucleotide structures. With the advent of more DNA-protein structure determinations, it is of interest to see how well the protein-refinement packages, PROLSQ and X-PLOR, and the small-molecule program, SHELXL93, are able to accommodate DNA. Comparisons are made between the dictionaries, weights and restraints used and the final models obtained from each program. Although the final R values, using all data in the resolution range 8.0-1.4 A, from PROLSQ (22.8%), SHELXL93 (R1 =21.7% after isotropic refinement) and X-PLOR (24.4%) are higher than the R value from the NUCLSQ refinement (21.2%), the root-mean-square deviations between the four final models are very small. Using this high-quality 8.0-1.4 A data set neither the dictionary nor the refinement program leave an imprint on the final fully refined complex. Likewise, the helical parameters and backbone conformation including sugar-puckering modes are not influenced by the refinement procedure used. Although a different number of water molecules is found in each refinement, varying from 62 (X-PLOR) to 86 (NUCLSQ), the first hydration sphere is well conserved in all four models.

An unprejudiced final model of the daunomycin-d(CGCGCG) complex was obtained at 1.1 Å resolution and at 100K after unrestrained SHELXL refinement, which was possible thanks to the high datato-parameter ratio and which provided us with true standard deviations on positions and distances. The structural pattern that emerges from the refinement proves that the sugar phosphate backbone is considerably more conformationally flexible than was previously observed [Biochemistry 1991, 30, 3812-3815; J. Biol. Chem. 1993, 268, 10095-10101]. All phosphates (as well as one sugar moiety) that are not rigidified by intercalation of the drug molecule are found to adopt two or more distinct conformations. Furthermore, the high quality of the data enabled us to find double conformations for several waters associated with the flexible phosphates or with flexible groups of the daunomycin molecule. These present findings suggest that the crystal lattice still allows for a certain conformational freedom of the DNA in the crystal. This freedom refers to the multiple conformations observed in the group of final models resulting from an NMR structure determination.

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