Abstract
The dodecamer d(CGCGAATTCGCG) was the first oligonucleotide to be crystallized as a B-DNA duplex. Its structure was analyzed in detail in the early 1980s. Here we show that, in the presence of Ca(2+), it crystallizes in a different way (R3 space group). The dodecamers form parallel columns of straight duplexes with ten base pairs in the B form. The terminal cytosines in each molecule are disordered, whereas the terminal guanines are placed in the minor groove of neighbor duplexes. The central GAATTC region is practically identical to that found in the classic structure of the same dodecamer crystallized in the P2(1)2(1)2(1) space group in the presence of Mg(2+) and spermine. Its structure is thus independent of the crystallization conditions which have been used.
Highlights
The first detailed structure of a DNA oligonucleotide in the B-form determined by single crystal x-ray diffraction methods was published in this journal in 1982 [1]
We discovered [14] that, in the presence of a high concentration of Ca2ϩ ion, this oligonucleotide could crystallize in the R3 space group
The results reported here for the d(CGCGAATTCGCG) dodecamer under very different packing constraints show that the central GAATTC sequence has a very stable conformation, very similar to that reported in the P212121 structure several years ago [1,2,3]
Summary
Crystallization—The crystal was grown by the vapor diffusion hanging drop method. The crystallization solution contained 0.4 mM dodecamer (NH4ϩ salt), 300 mM CaCl2, 10% MPD (2-methyl-2,4-pentanediol), and 20 mM cacodylic acid (pH 7.0). A high resolution (1.20 Å) dataset was collected with long exposure, and a second dataset at low resolution (2.65Å) was collected with short exposure to avoid saturation of some strong low resolution reflections that are important for the interpretation of the electron density map during the structural refinement Both datasets were processed with the program DENZO [15] and reduced and merged with the program SCALEPACK [15]. It was noticed during data processing that the diffraction of the crystal is rather anisotropic, which is probably because of the nature of crystal packing and the disordered terminal cytosine residues. It diffracts to 1.2 Å in the z direction but to a lower resolution in perpendicular directions.
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