Short CGG repeats were shown to form a duplex structure (20,21) and in the presence of potassium ions— G-tetraplexes (22–24). Thus, an RNA structure formed by CGG repeats is less clearly deﬁned than in the case of CUG repeats (18,19,25) and CAG repeats (19,26–28) for which crystal structures have been determined (29–31). In this study, we report three crystal structures of double-helical CGG repeats containing native and 8-Br modiﬁed guanosine residues. MATERIALS AND METHODS Synthesis, puriﬁcation and crystallization of oligoribonucleotides GCGGCGGC, GC(8-BrG)GCGGC and GC(8-BrG)GC GGCGGC oligomers were synthesized on an Applied Biosystems DNA/RNA synthesizer, using cyanoethyl phosphoramidite chemistry. Commercially available C and G phosphoramidites with 20-O-tetrbutyldimethylsilyl were used for the synthesis of RNA (Glen Research, Azco, Proligo). The phosphoramidite 8-Br guanosine was synthesized according to Proctor et al. (32). The details of deprotection and puriﬁcation of oligoribonucleotides were described previously (33). All crystals were grown by the hanging drop/vapour diffusion method at 19 C. A single crystal of GC(8-BrG)GCGGCGGC grew in 10 months. The reser- voir initially contained 10 mM MgCl2 50 mM Na cacody- late, pH 6.0 and 1.0 M Li2SO4. The crystallization drop initially contained 3 ml of RNA at 10 mg/ml and 1 mM MgCl2, and 3 ml of the reservoir solution. Crystals of GCGGCGGC grew in several days from the same solution as above, but involved crystal seeding and the starting RNA concentration of 2.4 mg/ml. The crystals grew as clusters of small needles which were then used for seeding. The seeded crystals grew from a similar solution but with the RNA concentration of 1.2 mg/ml. They appeared as single blocks but were in fact clusters of crystals that had to be separated. Crystals of GC(8-BrG)GCGGC grew in 2 months. The reservoir ini- tially contained 10 mM CaCl2, 0.2 M NH4Cl, 50 mM Tris– HCl at pH 8.5 and 30% w/v PEG 4000. The crystalliza- tion drop initially contained 2 ml of RNA at 10 mg/ml and 1 mM MgCl2, and 2 ml of the reservoir solution. X-ray data collection, structure solution and reﬁnement X-ray diffraction data were collected at 100 K: from GCG GCGGC on BL 14.2 beam line at the BESSY synchrotron (Berlin) to the resolution of 2.05 A˚ ; from GC(8-BrG)GCG GCGGC and GC(8-BrG)GCGGC on EMBL X11, DESY, Hamburg, to the resolution of 1.45 A˚ and 0.97 A˚, respectively. The crystals were cryoprotected by 20% glycerol (v/v) in the mother liquor. The data were integrated and scaled using the program suite DENZO/ SCALEPACK (34). The structure of GC(8-BrG)GCGGC was solved ﬁrst. SHELXD was used to identify the positions of the Br atoms by analysing the Patterson function based on the anomalous signal (35). The program identiﬁed two sites at least ﬁve times higher than any other peaks. SHELXE was Nucleic Acids Research, 2011, Vol. 39, No. 16 7309 used to identify the correct enantiomorph and to calculate initial phases (35), the calculated electron density map was uninterpretable in terms of an atomic model, but showed parallel columns of density indicating stacked RNA duplexes. DM was used for density modiﬁcation but the resulting maps were still uninterpretable (36). The phases from DM were used in a free-atom phase reﬁnement with ‘shaking’ of the model, using ARP/wARP (37). In a total of 100 cycles of adding/removing atoms with nine round of shaking interspersed, the R-factor/R-free statistics ini- tially remained apparently random but after 25 cycles started dropping and in the ﬁnal 50 cycles collapsed to 0.188/0.256. The free-atom electron density map showed the RNA atoms clearly resolved and a well-deﬁned solvent structure; the map looked very similar to the ﬁnal map for the reﬁned model. Both GC(8-BrG)GCGGCGGC and GCGGCGGC structures were solved by molecular re- placement using PHASER (38). The initial models were poor but sufﬁcient for the purpose of reﬁnement and model extension. The manual rebuilding and map inspec- tion were done using Coot (39). All three structures were reﬁned using Refmac5 (40) and Phenix (41). The ﬁnal model of [GC(8-BrG)GCGGC]2 was reﬁned without restraints and with anisotropic tem- perature factors. The last few cycles of the [GC(8-BrG)GC GGC]2 reﬁnement were performed using all data, including the Rfree set. The other two models, GCGGC GGC and GC(8-BrG)GCGGCGGC, were reﬁned using isotropic B-factors. Helical parameters were calculated using 3DNA (42). Sequence-independent measures were used based on vectors connecting the C10 atoms of the paired residues, to avoid computational artefacts arising from non-canonical base pairing. Program PBEQ-Solver (43) was used to calculate electrostatic potential map. All pictures were drawn using PyMOL v0.99rc6 (44). The coordinates of the crystallographic models have been deposited with the Protein Data Bank (PDB) with acces- sion codes 3R1C, 3R1D and 3R1E. RESULTS AND DISCUSSION The overall structures The RNA in all three crystal structures has the form of duplexes stacking end-to-end and forming bundles of parallel columns. The crystals of the native RNA contain 18 (GCGGCGGC)2 duplexes in the P1 unit cell. Each column consists of all the 18 independent duplexes stacked consecutively (Supplementary Figure 1). In the atomic resolution C2 structure, there is one [GC(8-BrG)GCGGC]2 duplex in the asymmetric unit. The other monoclinic crystal contains ﬁve crystallograph- ically independent RNA strands. They form three (GC(8-BrG)GCGGCGGC]2 duplexes, the third consisting of two symmetry-related strands. The native RNA and [GC(8-BrG)GCGGCGGC]2 structures contain sulphate ions from the crystallization medium, while [GC(8-BrG)GCGGC]2 crystals have Ca2+. All the ions interact in the major groove with the G–G pairs (details below). The ﬁnal models are summarized in Table 1.