3432 Nucleic Acids Research, 2005, Vol. 33, No. 10 Figure 5. Details of the DNA–DNA contact within the human NCP crystal. (A) Detailed stereo view of the DNA–DNA contact shown in Figure 4B and C. (B) Detailed stereo view of the DNA–cation–DNA contact shown in Figure 4D. All of the atoms of the DNA duplexes are shown as normal sticks, where the phosphorus, oxygen and nitrogen atoms are colored cyan, red and blue, respectively. The Mn2+ ions and the water molecules are depicted by black and red balls, respectively. Broken lines and distances between two atoms show possible interactions through water molecules. Numbers presented for each base pair are the same as those in Figure 2A. This inter-particle contact may simultaneously generate the sequential 1 bp shift, which ranges from SHL 5 to 2 (Figure 4A, black arrows). This alteration of the human DNA path expands by 2 s between two DNA duplexes, wrapping around the histone octamer, as compared with that of Xla-NCP (Figure 3C). The resultant space accommodates a short seg- ment of a DNA strand in the adjacent NCP, because of the tighter crystal packing (Figure 4A, green circle). This partial insertion of DNA also generates the tighter twist in the DNA path at SHL 5 of the neighboring NCP (Figure 4D) and another consecutive 1 bp shift from SHL 5 to 7 at the terminus (Figure 4A, black arrow). In detail, it is likely that the intermolecular contacts occur through water-mediate inter- actions between the Mn2+ ion bound to the minor groove and the phosphate group at the location 53 and between the two phosphate groups at the location 38 and 53 (Figure 5B). The DNA–cation–DNA contact may modify the DNA structure by the reciprocal ﬁt of the sugar–phosphate backbone into the minor groove. Although experimental data are unavailable at the moment, a similar change in the DNA path may take place in vivo, coupled with the contacts between NCPs in the compacted state of human chromatin. Effects of histone sequence differences The amino acids of human histones that differ from those of X.laevis homologs are located within the common octameric core structure. Notably, only Ala116 in H2A lies on the molecular surface, and it is too far away to interact with the neighboring NCP. Ser38 and Val39 in H2B are close to the histone–DNA contact region, but do not appear to parti- cipate in the contact. Then, what causes the alteration of the DNA path and the uniquely compact packing in human NCPs? It is likely that Lys30, Arg31 and Ser32 in the H2B tail (triplet residues: Arg30, Lys31 and Thr32 in Xla-H2B) perturb the DNA path. In fact, the superposition between human and X.laevis NCPs revealed that human Arg 31 structurally clashes with the DNA backbone in Xla-NCP (SHL 5; Figure 3A, stereo view). In the symmetric part of the human H2B tail (SHL 5; Figure 3B, stereo view), the DNA path is also altered by the steric hindrance between the DNA and the H2B tail, which has a different conformation from that of the other H2B tail. Notably, the corresponding triplets in the H2B tail (Lys30, Arg31 and Ser32 in human; Arg30, Lys31 and Thr32 in X.laevis; Ser32, Lys33 and Val34 in yeast; and Lys30,