7310 Nucleic Acids Research, 2011, Vol. 39, No. 16 Table 1. Summary of the X-ray data and model reﬁnement for (GCGGCGGC)2, [GC(8-BrG)GCGGCGGC]2 and [GC(8-BrG)GCGGC]2 Crystal Beam line Wavelength (A˚ ) Space group Cell parameters Resolution range (A˚ ) Mosaicity ( ) Exposure time per image (s) Rmergeb <I/s(I)> Completeness (%) No. unique reﬂections Overall multiplicity Reﬂections > 3s (%) B-factor from Wilson plot (A˚ 2) Rworkc Rfree No. RNA atoms Ions No. water molecules Other solvent r.m.s. deviation from ideal values Bond lengths (A˚ ) Bond angles ( ) PDB code GCGGCGGC BESSY BL 14.2 0.9200 P1 a = 39.7, b = 76.9, c = 85.4 A˚, = 90.0, = 88.6, = 77.3 20.0–2.05 (2.09–2.05)a 0.3 5–10 0.093 (0.488) 17 (2.8) 98.4 (97.5) 60 328 4.4 (3.5) 71 (40) 33 21.56 25.71 6304 7 SO42 524 – 0.005 1.145 3R1C GC(8-BrG)GCGGC EMBL-X11 0.8126 C2 a =50.7, b = 22.5, c = 44.2 A˚, = 117.8 20.0–0.97 (0.99–0.97) 1 40 0.132 (0.934) 9 (2.5) 95.7 (93.4) 24 974 5.4 (4.7) 77 (29) 7.8 13.66 – 370 2 Ca2+ 103 1 glycerol 0.011 1.746 3R1E GC(8-BrG)GCGGCGGC EMBL-X11 0.8126 C2 a = 118.6, b = 28.6, c = 61.8 A˚, = 118.0 20.0–1.45 (1.47–1.45) 0.3 50 0.112 (0.620) 10 (3.3) 99.8 (100.0) 32 941 7.5 (6.3) 77 (50) 21.2 23.21 27.02 1617 8 SO42 207 – 0.006 1.282 3R1D aValues in brackets are for the highest resolution shell. bRmerge = hkl i |Ii(hkl) <I(hkl)>|/ hkl i Ii(hkl), where Ii(hkl) and <I(hkl)> are the observed individual and mean intensities of a reﬂection with indices hkl, respectively, i is the sum over the individual measurements of a reﬂection with indices hkl and hkl is the sum over all reﬂections. cRfree was calculated using 5% of the total reﬂections chosen randomly and omitted from the reﬁnement. Non-canonical G–G pairing and its consequences on the duplex Despite the presence of the non-canonical base pairs, the helices have a typical A-form, with values of helical twist in the range 30–32 for all the duplexes, the sugar pucker C30-endo or, in some cases, C20-exo and Zp values of 2.63 ± 0.22 A˚ (for the A-form it should be more than 1.5 A˚ ). The helices contain C–G and G–C base pairs with typical Watson–Crick interactions. Between them are the non-canonical G–G pairs in which one guanosine is always in the syn conformation while the other is anti (Figure 1), experimentally shown to be the preferred ar- rangement in double helical context (45–47). The syn-anti geometry of this base pair can be described as G/G cis Watson–Crick/Hoogsteen according to the nomenclature proposed by Leontis and Westhof (48). There are two hydrogen bonds between the guanosine residues: carbonyl oxygen is bonded to N1H, and N7 to the exo- amino group. All the H-bond distances are in the range 2.6–3.3 A˚ . The conformation of the G(syn) residue is add- itionally stabilized by a hydrogen bond between the exo-amino function and its phosphate oxygen atom (3.0 A˚ ). The syn-anti arrangement avoids the steric clash between the two bulky guanines within the helical struc- ture. This is evident from the C10–C10 distances between the paired residues: 11.3 ± 0.1 A˚ for G–G, compared with 10.7 ± 0.2 A˚ for the canonical C–G pairs. The angle of Figure 1. Non-canonical G(syn)–G(anti) pair. Hydrogen bonds are drawn with dashed lines. Solid line connecting C10 atoms gives a measure of strand separation. Angles are marked (see text). the glycosidic bond with the line connecting the C10 atoms of each pair is 33 ± 4 for the G(syn) residues and 64 ± 3 for G(anti), compared with 54 ± 3 for the other residues. This means that the G(syn) is shifted towards the minor groove and the G(anti) towards to major groove, which optimizes the H-bonding interactions between the Hoogsteen and Watson–Crick edges, while avoiding the clash between the carbonyl oxygen atoms (Figure 1). The G(syn) residues also show unusual a and g backbone torsion angles. The a angle, representing a rotation about the P-O50 bond, is +ac, +ap, or ap (in one case) instead of the typical sc. The angles range 107–182 with the average value of 142 , almost half a turn from the mean sc value of 60 . The g angle, about the C50-C40 bond, is +ap or ap in the G(syn)