C.R. Clapier et al. Finally, the nucleosome is involved in major cellular reg- ulatory mechanisms related to cell cycle and aging, cell differentiation, and cellular reprogramming,9 and plays a critical role in viral infection10 and cancer.11 Structural studies of the nucleosome over the last 20 years have painted an increasingly accurate picture of how the nucleosome accomplishes its packaging and regulatory roles. Initial low-resolution studies12–14 followed by the crystal structure of the histone octamer15 elucidated the overall architecture of the NCP. The structure of an NCP (Xla-NCP146) composed of recombinant Xenopus laevis histones and a 146-bp palindromic fragment of human a- satellite DNA revealed details of the DNA structure and its interactions with the histones.16 A higher resolution struc- ture (Xla-NCP147) using a related 147-bp DNA fragment allowed for a detailed analysis of the DNA conformation, solvent structure, and interactions with ions.17–19 Struc- tures of a Xenopus NCP containing the histone variant H2A.Z20 or macroH2A21 and of NCPs comprising chicken,22 yeast,23 and human histones24 have brought additional functional and evolutionary insights. To extend this analysis, we determined the crystal structure of the NCP from Drosophila melanogaster, the first from an invertebrate species. Drosophila histones share a high degree of sequence identity with those of Xenopus, ranging from 83% and 89% identity for H2B and H2A, respectively, to 99% for H3 and H4. Most of these changes localize to histone tail residues that are dis- ordered in the available NCP crystal structures. However, a substantial number involve structured histone residues. We compare the Drosophila and Xenopus NCP structures and focus particularly on histone residues that have diverged between these species. MATERIALS AND METHODS Crystallization NCPs were prepared from recombinant D. melanogaster histones and a 147 bp palindromic DNA fragment derived from human a-satellite DNA, as described previously.25 Crystallization trials were carried out by the hanging drop vapor-diffusion technique at 48C by equilibrating a drop- let containing 3 mg/mL Dm-NCP147, 80–85 mM MnCl2, 50–80 mM KCl, and 20 mM potassium cacodylate (pH 6.0) against a reservoir solution containing of 40– 42.5 mM MnCl2, 25–40 mM KCl, and 20 mM potassium cacodylate (pH 6.0). To improve diffraction quality, crys- tals were soaked overnight in the reservoir solution sup- plemented with 24% (v/v) 2-methyl-2,4-pentanediol as cryoprotectant and flash-cooled in liquid nitrogen. Crystallography Table I Data and Refinement Statistics Data collection Space group Cell parameters () ESRF beamline Resolution Overall () Outer shell () Completeness (%)a No. reflections, total No. reflections, unique Redundancy Rsym (%)b I/r(I) Structure Refinement Rcryst/Rfree (%)c No. of atoms Protein DNA Water Ions R.m.s.d.bond lengths ()/angles ( 8) Residues in Ramachandran plot (%) Most favored/allowed Mean B-factors ( 2) Protein/DNA P212121 a 5 106.0, b 5 182.0, c 5 109.4 ID14-3 30–2.45 2.5–2.45 94.6 (84.6) 234,968 (10,078) 74,234 (3871) 3.2 (2.6) 6.2 (48.1) 12.4 (1.7) 22.9/26.2 6103 6021 88 18 0.009/1.2 94.1/5.9 51.9/102.2 aValues in parentheses are those for the outer resolution shell. bRsym 5 Shkl|Ihkl 2 <I>|/ShklIhkl, where Ihkl is the measured intensity of reflec- tions with indices hkl. cRcryst 5 Shkl|Fo 2 Fc|/ShklFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is equal to Rcryst for a randomly selected 5% subset of reflections not used in the refinement. suite.27 Crystals obtained using described conditions17 were isomorphous to the Xla-NCP147 crystal form (Ta- ble I). The Xla-NCP147 structure (pdb id 1KX5) minus the N-terminal histone tail residues was used as a starting model. Positioning this model into the Dm-NCP147 unit cell resulted in a crystallographic R-factor of 0.40, which dropped to 0.32 upon rigid body refinement using CNS.28 A further round of restrained coordinate and B- factor refinement reduced this to 0.276 (Rfree 5 0.301). Differences between the Drosophila and Xenopus struc- tures were readily apparent in a 2Fo 2 Fc map calculated using phase information from the Xla-NCP147 atomic coordinates. Iterative rounds of manual model building using O29 and CNS refinement were carried out to incorporate amino acid substitutions, ions, and water molecules, and to rebuild the histone tails. The structure was refined at 2.45 A˚ to a final crystallographic R-factor of 0.229 (Rfree 5 0.262) and good geometry. RESULTS AND DISCUSSION Structural conservation between the Xenopus and Drosophila NCPs Diffraction data were collected at ESRF beamline ID14-3 (k 5 0.931 A˚ ) on a MAR CCD detector and processed with XDS26 and programs of the CCP4 As expected, Dm-NCP147 and Xla-NCP147 share a high degree of structural similarity. The histone octamers of the two particles superimpose with an overall root- 2 PROTEINS