Toward higher resolution nucleosome structure

Several steps were needed to determine the structure of the core particle to higher resolution (Fig. Id). The X-ray phases of the low-resolution models were insufficient to extend the structure to higher resolution, since the resolution of the early models of the NCP was severely limited by disorder in the crystals. The disorder was presumed to derive from both the random sequences of the DNA and from heterogeneity of the histone proteins caused by variability in post-translational modification of the native proteins. One strategy for developing an atomic position model of the NCP was to develop a high-resolution structure of the histone core. This structure could then be used with molecular replacement techniques to determine the histone core within the NCP and subsequently identify the DNA in difference Fourier electron density maps. 

An alternative approach to higher resolution nucleosome structure was to solve the complete NCP structure by increasing the diffracting resolution of 

NCP crystals. There were two facets to this approach. First, it was necessary to reconstitute NCPs from a defined sequence DNA that phased precisely on the histone core to circumvent the random sequence disorder. It was obvious that the DNA was important for the quality of the diffraction from NCP crystals but the role of histone heterogeneity was not so clear. Heavy atom derivatives (i.e., electron rich elements bound in specific positions on the proteins) were not readily prepared by standard soaking experiments, due to a paucity of binding sites. Hence, it was necessary to selectively mutate amino acid residues in the histones to create binding sites for heavy atoms. 

At Oak Ridge, the focus was to develop specific-sequence DNA to improve the diffraction quality of NCP crystals. The positioning of the DNA on the histone core has to be precise so that all the NCPs are identical. A project was undertaken to understand the DNA sequence effects on nucleosome phasing [25]. Second, a DNA palindrome was developed to extend the two-fold symmetry of the histone core to the DNA. The objective was to eliminate the two-fold disorder caused by the indeterminacy of packing of an asymmetric particle into the crystal lattice. A palindrome based on one-half of the primary candidate sequence was constructed and methods were developed to produce the palindrome fragment in large quantities for reconstitution of NCPs. 

After the a-satellite DNA palindrome was constructed and cloned, the task remained to produce it in large quantities. To do this multiple copies of the halfpalindrome fragment were cloned into a vector [26]. The half-palindrome fragment 

---