sequence differences between these histones. However, the path of the nucleosomal DNA in human NCP is different from that within Xla-NCP (11), in spite of their identical 146 bp sequences. MATERIALS AND METHODS Expression, purification and reconstitution of human NCP The four core histone genes, forming a cluster (22,23), were ampliﬁed by PCR using genomic DNA obtained from HeLa cells. Vectors for the expression of all four recombinant human core histone proteins were constructed by inserting the gene fragments into pET-22b (Novagen). The histone genes in each of the resulting plasmids were sequenced to conﬁrm their integrity. The histones were overexpressed in BL21 (DE3) codonplus RIL (Stratagene) and were puriﬁed using the pro- tocols published previously (24,25). The histone proteins were refolded into a histone octamer. The pUC-based vector, a kind gift from Dr K. Luger, contains one-half of a 146 bp palindromic DNA fragment derived from a human a-satellite region (12). The 146 bp palindromic DNA fragment was con- structed from this vector and puriﬁed as described previously (24,25). The histone octamers were reconstituted into NCPs using the 146 bp palindromic DNA fragment (24,25). Milligram amounts of human NCPs were subjected to heat shifting, followed by subsequent puriﬁcation using anion exchange chromatography on a Mini Q column (Amersham Biosciences). Crystallographic procedures Crystals of a human NCP were obtained by vapor diffusion, at a protein concentration of 4 mg/ml with 55–58 mM KCl, 70–80 mM MnCl2 and 20 mM potassium cacodylate, pH 6.0 in a drop equilibrated against 35 mM KCl, 40–45 mM MnCl2 and 20 mM potassium cacodylate, pH 6.0. The crystals were transferred into 37 mM MnCl2, 40 mM KCl, 20 mM potassium cacodylate, pH 6.0, 28% (v/v) 2-methyl-2,4-pentanediol and 2% (w/v) trehalose for cryoprotection and improvement of the diffraction resolution. Crystals were ﬂash-frozen in liquid nitrogen directly from the cryoprotectant condition. X-ray diffraction data were collected at the synchrotron radiation source at BL41XU (SPring-8, Hyogo, Japan), at 100 K using a nitrogen stream. Processing of the diffraction images and scaling of the integrated intensities were carried out using the program HKL2000 (26). Molecular replacement, with the PDB entry 1AOI (12) as the search model, was used to obtain the initial phases via Amore (27). Reﬁnement was performed using CNS (28), and model building into the 2Fo Fc and Fo Fc electron density maps was carried out by the use of program O (29). Mn2+ and Cl ions were initially located in the Fo Fc electron density maps as solvent peaks (>3s) with the surrounding molecular environments accommodating one or more potential hydrogen bonds to the peak. These ions were identiﬁed as described in a following section. DNA base-pair-step parameters were cal- culated using the program curves (30). Graphic ﬁgures were prepared using the program UCSF chimera (31). The coordin- ates and structure factors have been deposited in the PDB under ID codes 2CV5. Nucleic Acids Research, 2005, Vol. 33, No. 10 3425 RESULTS AND DISCUSSION Overall structure A human NCP was crystallized under conditions similar to those previously published for the crystal structure of a Xla-NCP (11,12). The human crystal showed the same ortho- rhombic space group as that of the X.laevis crystal (Table 1). However, the unit cell parameters were substantially different from those of Xla-NCP, with an exchange of the b- and c-axes and a 13% reduction of the unit cell volume, implying a denser packing than that in the Xla-NCP crystal (11,12). The crystal structure was determined by the molecular replacement method, using the previously published Xla-NCP structure (PDB entry 1AOI) (12) as a search model. Data collection and ﬁnal reﬁnement statistics are summarized in Table 1. Sequence differences between the human and X.laevis histones were discernible in the original 2Fo Fc electron density map (data not shown). Amino acid sequence alignments between the human and X.laevis histones revealed that H2A, H2B and H3 are slightly divergent (92.1, 93.6 and 98.5% identity, respectively), whereas H4 is identical between the two species. This degree of sequence divergence is comparable with that between the primary structures of the human and chicken histones. In agreement with this minor difference, the overall structure of a human NCP (Figure 1) is very similar to those of Xla-NCP Table 1. Data and refinement statistics Data collection statistics Wavelength (A˚ ) Space group Unit cell parameters (A˚ ) No. of unique reflections Resolution (A˚ )a Rmergea,b Completeness (%)a I/sa Refinement statistics Number of protein residues in the final modelc Number of base pairs in the DNA Number of water molecules Number of Mn ions Number of Cl ions Total number of atoms in the final model Rwork/Rfree (%)d R.m.s.d from ideality Bond (A˚ ) Angles ( ) Average B-factors (A˚ 2) Protein DNA Solvent 1.0 P212121 a = 99.6, b = 108.4, c = 169.4 61 700 50–2.50 (2.59–2.50) 7.8 (49.5) 96.0 (84.5) 28.8 (3.3) 761 146 430 9 4 12 475 22.38/27.69 0.0062 1.14 28.91 51.80 33.36 aValues in the parentheses are for the final shell. bRmerge ¼ P jIhklh Ihklij= P Ihkl, where Ihkl is the intensity measurement for the reflection with indices hkl, and hIhkli is the mean intensity for multiply recorded reflections. cResidues included in each histone subunit: H3: 38–134, H30: 37–135, H4: 25–102, H40: 18–102, H2A: 11–118, H2A0: 15–118, H2B: 30–125, H2B0: 31–124. The remaining portions of the histone tails were too disordered to be included in the final model. dRwork‚ free ¼ P jjFobsjj Fcalcjj=jFobsj, where the R-factors were calculated using the working and free reflection sets, respectively. The free reflections comprise a random 10% of the data reserved for unbiased cross-validation throughout the refinement.