Structure of the Drosophila Nucleosome Core Particle Table II Comparison of Dm-NCP147 and Xla-NCP147 Structures All structured residues Terminal res. excludedd RMSD () a Excluding terminic 0.33 (0.64) 0.35 (0.84) 0.28 (0.88) 0.33 (0.68) 0.15 (0.64) 0.17 (0.58) 0.15 (0.43) 0.18 (0.57) 0.30 (0.70) 0.32 (0.59) 118–119 12–13 28–29 29–29 –– 135 23–24, 102 15–19 All above All above Seq. identity (%)b Overall/structured 88.7/91.6 82.7/93.6 99.2/99.0 99.0/100 aRMSD values for the backbone Ca and DNA phosphate atoms; values in parentheses are for all atoms including side chains. bPercent sequence identity between Drosophila and Xenopus histones for all residues, and for residues present in the crystallographic model. cRMSD values in which structurally most divergent N- and/or C-terminal residues are excluded from the alignment. dN- and/or C-terminal residues excluded from the alignment. H2A H2A0 H2B H2B0 H3 H30 H4 H40 Histone octamer DNA NCP 0.36 (0.65) 0.67 (1.18) 0.48 (1.02) 0.49 (0.77) 0.15 (0.64) 0.26 (0.65) 0.78 (0.95) 0.93 (1.72) 0.58 (1.00) 0.37 (0.34) 0.54 (0.83) mean-squares deviation (rmsd) of 0.58 A˚ for backbone Ca atoms, and 1.00 A˚ for all atoms including side chains (Table II). These values are approximately half those obtained upon alignment of the yeast and Xenopus octamers23, consistent with the notion that structural divergence recapitulates phylogeny.30–33 The individual histone Ca backbones can be aligned with rmsd values of 0.15–0.93 A˚ . However, these values reduce to 0.15– 0.35 A˚ upon exclusion of a small number of N- or C-ter- minal residues, where the most significant differences occur. These reduced values correlate well with degree of sequence conservation, the more divergent H2A and H2B histones showing larger rmsd values than the nearly invariant H3 and H4 histones (Table II). The conformation of the DNA is essentially identical in the two structures (rmsd 5 0.34 A˚ for all atoms). Unlike the structure of human NCP146, in which the DNA at three superhelix axis locations (SHLs) is shifted relative to Xla- NCP146,24 the DNA in Dm-NCP147 remains in register with that of Xla-NCP147.17 This is likely a reflection of the higher degree of order generally observed in NCP147 com- pared to NCP146, irrespective of the source of histones. As in previous NCP structures, a plot of B-factor versus base pair shows an oscillating pattern, with minima (40–80 A˚ 2) where the DNA contacts histones, and maxima (80–160 A˚ ) at intermediate positions. The manganese and chloride ions identified in Xla-NCP14718 are all preserved in our Drosophila structure. The entire structure can be superim- posed onto that of Xla-NCP147 with an rmsd of 0.82 A˚ for all protein and DNA atoms, underscoring the high degree of tertiary and quaternary structure conservation. Differences in the histone tails A comparison of the Dm- and Xla-NCP147 structures reveals slight differences in the histone tail regions. These probably reflect the inherent structural disorder of the tails, but may also reflect sequence differences (Fig. 1, residues highlighted in pink. Sequence numbering throughout this paper is that of Xenopus, which is identi- cal to the Drosophila numbering except for H2A). More specifically, in Xla-NCP147, H2A0 residue Lys13 inserts into the minor groove to hydrogen bond with Thy 45 in SHL 4 [Fig. 2(A,B)]. In the Drosophila structure, the cor- responding Lys residue points more toward the solvent, interacting with the adjacent DNA phosphate group. A few residues away, the Drosophila and Xenopus H2A sequences diverge at two positions, with Drosophila resi- dues Ser and Asn replacing Thr16 and Ser19, respectively. In the Xenopus NCP, Ser19 hydrogen bonds to the back- bone amide of Thr16, one helical turn away, with the lat- ter in van der Waals contact with the DNA phosphate backbone [Fig. 2(B)]. While the DNA contact is pre- served in Dm-NCP147, the intrahelical hydrogen bond is not, its loss compensated by a hydrogen bond gained between the Ser16 and Asn19 side chains [Fig. 2(B)]. In the H2B0 chain of Xla-NCP147 (and -NCP146), res- idue Thr29 hydrogen bonds with the DNA phosphate backbone (at nucleotide position 30 of chain J) [Fig. 2(C,D)]. In Dm-NCP147, the corresponding Lys29 resi- due interacts with a phosphate group on the complemen- tary strand (position 26 of chain I). The preceding Arg28 residue adopts a similar orientation as the Xenopus Lys28 residue, but inserts more deeply into the minor groove, interacting with the Cyt-49 (chain J) and Thy-50 (chain I). An Arg side chain in a minor groove is a recurrent motif, observed in both the tail and core regions of the various histone chains.16 Residues 28 and 29 in the Dm- NCP147 H2B chain are located approximately as in H2B0, but are considerably more disordered. Such varia- tions between otherwise identical chains highlight the structural plasticity of the histone tails. PROTEINS 3