Direct-methods determination of an RNA/DNA hybrid decamer at 1.15 A resolution

For the first time, ab initio direct methods have been used to solve the crystal structure of an RNA/DNA hybrid decamer. The RNA/DNA sequence corresponds to the leftmost two-thirds of the polypurine tract (PPT), the primer for second-strand DNA synthesis by HIV-1 reverse transcriptase (RT). Direct methods using Shake-and-Bake (SnB) yielded solutions for the RNA/DNA decamer molecule using 1.15 A data, which is just on the resolution edge of what might work with direct methods. Atomic positions for 96% of the entire molecule, containing 514 non-H atoms including three Ca(2+) ions, were easily interpreted from a Fourier map based on the 'Shake-and-Bake' minimal function and CROQUE phase-refinement program. Only six atoms, primarily in the sugar linkage, were missing in this Fourier map. At present, the R factor of the model is 0.143 (R(free) = 0.186) for the 562 non-H atom sites located. The conformation of the RNA/DNA helix is A-form, with a typical A-helix minor-groove width. This paper presents the methodology used in solving this structure.