Lys31 and Ser32 in chicken) occupy identical positions in the 3D structures, although the equivalent H2B segment does not affect the nucleosomal DNA path in either chicken (13) or yeast (14). However, the triplet residues are highly conserved among the human H2B variants (17). In addition, it has been shown that the deletion of the yeast H2B tail segment, con- taining the triplet residues, signiﬁcantly decreases plasmid superhelical density, suggesting a release from the DNA con- straints within the nucleosome (39). It is thus possible that the conformational change in the histone tail locally perturbs the DNA path, although it is unlikely to generate the entire alteration around SHL 5 to 2 and 5 to 7 (Figure 3). Ion-binding sites Speciﬁc ion-binding sites in the protein and DNA structures were identiﬁed from the residual peaks (>3s) in the Fo Fc difference maps, with information on the composition and the metal ion coordination geometry. This approach allowed us to identify 13 bound ions in a human NCP. Five are bound to the protein alone and eight are associated with the DNA. One peak corresponds to a Mn2+ ion bound between particles in the Xla-NCP crystal structure (11). Therefore, this peak was iden- tiﬁed as a Mn2+ ion, which is coordinated by the carboxylic oxygen of H3-D77 from one particle, the peptide carbonyl oxygen of H2B-V45 from the other and two water molecules. Four chloride ions were identiﬁed, as two peaks of the H3-a3 helices near the center of the DNA and two peaks of the H2B-a3 helix and H2A-a2 helix, in agreement with the Xla-NCP structure at 1.9 s resolution (PDB code 1KX5) (11). Eight peaks observed in the DNA region could be identiﬁed as Mn2+ ions, because no other DNA-bound metal ions were present in the crystallization solution, except for potassium ions. In addition, the distances of the metal ions to the ligands ranged from 2.24 to 2.89 s, which are much shorter than the minimal K+ to ligand distance of 2.9 s observed in oligo- nucleotide DNA (40). The seven Mn2+ ions bound in the NCP-DNA major groove interacted exclusively with the N7 and O6 atoms of the guanines at GG, GC and GA steps, either directly or via the water molecules in their coordination shell. Of these sites, six at the base pair locations of 60, 47, 3, 35, 48 and 61 were the same binding sites (Figure 2A) as those in the 2.0 s structure of Xla-NCP (PDB code 1KX3) (11). The single remaining Mn2+ binding site, at the base pair location of 5, was observed in the 1.9 s structure of Xla-NCP (PDB code 1KX5) (11,15). However, in comparison with Xla-NCP, the positions of the two Mn2+ ions (at 47 and 61) were shifted, presumably because of coupling with the alteration of the DNA path. The four Mn2+ binding sites at 34, 26, 27 and 65, which were fully occupied by the metal in Xla-NCP (11), were vacant in the human NCP structure. Three of them (at 34, 26 and 65) are located in the altered DNA path (Figure 2A). Metal ions bound to DNA generally contribute to the stabilization of the preexisting conformation (41,42). Consistently, in the region of 37 to 18, which lacks Mn2+ ions in human NCP, the DNA phosphates have substantially high B-factors (Figure 2A). This instability may be connected with the altera- tion of the DNA path. A unique Mn2+ ion binding site was found in the minor groove at only one location 40 in the entire human NCP Nucleic Acids Research, 2005, Vol. 33, No. 10 3433 structure (Figure 2A). A similar Mn2+ binding mode in the minor groove was observed at different base pair position 13 in the Xla-NCP structure (11,15). This Mn2+ ion in an Xla-NCP is bound between the minor groove and the H2B-aC helix belonging to the neighboring NCP (15). As described above, the Mn2+ ion in a human NCP is very close to the phosphate group at the location 53 between adjacent NCPs (Figure 5B). Internucleosomal histone– cation–DNA or DNA–cation–DNA interactions, as observed previously (15,43), may mediate chromatin compaction. This DNA–cation–DNA contact probably contributes to the tight crystal packing of a human NCP. CONCLUSION We have determined the crystal structure of a human NCP at 2.5 s resolution. Our ﬁndings conﬁrm that the overall NCP structures are conserved between human and other eukaryotes, in spite of the considerable differences in the chromatin organ- ization between the eukaryotes (32–34). However, the path of the nucleosomal DNA exhibits a remarkable alteration in the human NCP crystal structure. This alteration appears to be caused by a combination of several factors, such as the con- formation of a H2B tail, the conversion of metal binding sites and the different crystal packing, suggesting that the nucle- osomal DNA conformation is more ﬂexible and sensitive to the environment than we expected. The extensive alteration of the human DNA path induces the space expansion between two DNA duplexes within a single NCP structure. The post-translational modiﬁcation of the his- tone tail may additionally contribute to the further expansion. It is intriguing to envisage that the resultant space is linked to interactions with some activators and chromatin remodeling factors. The demonstration of this hypothesis awaits further studies, such as the crystal structure determinations of NCPs complexed with some factors. However, it was previously proposed that nucleosome repositioning by 2 or 3 bp contrib- utes to a subtle conformational change, but highly effective mechanism for gene regulation (44). Furthermore, although the nucleosomes are still packaged in the ﬁrst event of the transcriptional activation, the chromatin is permissive for the association of the activator HNF-1a, TBP and TFIIB (21,45). Thus, the DNA path alteration, accompanied by 1 bp shift, may be important in the transcriptional mechanism recruiting some factors, such as the activators, transcription factors and chromatin remodeling factors, onto NCPs. ACKNOWLEDGEMENTS The authors thank Drs Takuji Oyama and Daisuke Tsuchiya for their support in X-ray crystallography and helpful discus- sions, and Dr Masahide Kawamoto for data collection at the SPring-8. The authors acknowledge Dr Toshiyuki Shimizu for helpful discussions about the DNA base-pair-step parameters and Dr Noriyuki Suka for helpful discussions. The authors are grateful to Dr Karolin Luger for the kind gift of the pUC-based vector, which generated the 146 bp human a-satellite DNA. This work was partly supported by a research grant endorsed by the Japan New Energy and Industrial Technology Development Organization (NEDO). Funding to pay the Open Access