Structure calculations

4.4.1 Structure calculation of d3'-EBS1*

NOE distances were estimated from the integrated peak volumes obtained from the 2D [1H,1H]-NOESY spectrum that was acquired at 293 K with a mixing time of 250 ms. Distances were calibrated using the CALIBA macro in DYANA.(397) The NOEs were grouped into four categories, corresponding to strong (1.8 – 3 Å), medium (1.8 – 4.5 Å), weak (3.0 – 6.0 Å), and very weak (4.0 – 7.0 Å) (Appendix 6). NOEs obtained from 2D [1H,1H]-NOESY crosspeaks in 90% H2O/10% D2O at 278 K were qualitatively assigned as strong, medium,

weak, or very weak (Appendix 6).

Based on 1D 31P NMR spectra, the torsion angles α and ζ were set to exclude the trans- range (except for G1, which was left unconstrained, as well as C29, where only α was

constrained) (Appendix 8).(121) Sugar pucker restraints were included based on 2D [1H,1H]- TOCSY experiments with a mixing time of 45 ms.(34) G1, A10 to A20, and C29 were left unconstrained. The other backbone torsional angles (β, γ, ε) were set to standard A-form values in the helical region of the structure (G2-U9, G21-C28). Based on the intranucleotide H1' to aromatic NOEs of a 60 ms NOESY spectrum the torsion angle χ was restrained to anti (–160 ± 20°) for all residues. Additional H-bond restraints were added for base pairs whose existence was proven by 1H-1H crosspeaks across the helix in the imino region and by HN- crosscorrelations from the 2D JNN HNN-COSY spectrum.(118)

200 initial structures were calculated with CNS 1.2 from an extended structure with random initial velocities using NOE distance, dihedral and H-bond restraints.(119) The structures were subsequently refined with the introduction of RDCs in XPLOR-NIH (Appendix 7).(120) The RDCs were measured using Sparky by determining the difference between 1H-13C and 1H-15N for isotropic and partially aligned samples. The Da and R values

of the alignment tensor were estimated using PALES(323,324) and further refined by manual gridsearch.(398) The final Da was set to –41 Hz and R to 0.3. Structures were refined by

cooling from 2000 K to 100 K. 500 steps of energy minimization using the Powell algorithm followed simulated annealing. For comparison 200 structures each were calculated with and without the introduction of RDCs. After refinement, the structures were evaluated for convergence. Acceptance criteria were low overall energies and no significant NOE (>0.2 Å) or dihedral (>5°) violations. The twenty lowest-energy structures out of 200 calculated were visualized and analyzed using MOLMOL.(29) Six additional structures of low overall energy also satisfied all RDC and NOE restraints, but these were discarded owing to antiparallel alignment: that is, RDC restraints can be satisfied either by A-form geometry or by antiparallel helical orientations that are rotated 180° about the order tensor frame Sxx and

Syy.(399)

4.4.2 Structure calculation of d3'-EBS1*·IBS1*

NOE distances were estimated from the integrated peak volumes obtained from the 2D [1H,1H]-NOESY spectrum that was acquired at 298 K with a mixing time of 250 ms. Distances were calibrated using the CALIBA macro in DYANA.(397) The NOEs were grouped into four categories, corresponding to strong (1.8 – 3 Å), medium (1.8 – 4.5 Å), weak (3.0 – 6.0 Å), and very weak (4.0 – 7.0 Å) (Appendix 17). NOEs obtained from 2D [1H,1H]-NOESY

Materials and Methods 167

crosspeaks in 90% H2O/10% D2O at 278 K were qualitatively assigned as strong, medium,

weak, or very weak (Appendix 17).

Based on 1D 31P NMR spectra, the torsion angles α and ζ were set to exclude the trans- range (except for G1, which was left unconstrained, as well as C29 and C65, where only α was constrained) (Appendix 19).(121) Based on TOCSY experiments with a 45-ms mixing time, nucleotides with strong H1'-H2' and H1'-H3' crosspeaks were restrained to S-type range (δ = 145 ± 20°) (A10, U11, and U12), those with absent H1'-H2' crosspeaks to N-type range (δ = 85 ± 20°), and nucleotides with intermediate crosspeak intensities (G1, C29, and C65) were left unconstrained.(34) The other backbone torsional angles (β, γ, ε) were set to standard A-form values in the helical region of the structure (G2-U9, G21-C28, G13-G19, and C59- U64). Based on the intranucleotide H1' to aromatic NOEs of a 60 ms NOESY spectrum the torsion angle χ was restrained to anti (–160 ± 20°) for all residues. Additional H-bond restraints were added for base pairs whose existence was proven by 1H-1H crosspeaks across the helix in the imino region and by HN-crosscorrelations from the 2D JNN HNN-COSY

spectrum.(118)

200 initial structures were calculated with CNS 1.2 from an extended structure with random initial velocities using NOE distance, dihedral and H-bond restraints.(119) The structures were subsequently refined with the introduction of RDCs in XPLOR-NIH (Appendix 18).(120) The RDCs were measured using Sparky by determining the difference between 1H-13C and 1H-15N for isotropic and partially aligned samples. The Da and R values

of the alignment tensor were estimated using PALES(323,324) and further refined by manual gridsearch.(398) The final Da was set to –40 Hz and R to 0.1. Structures were refined by

cooling from 2000 K to 100 K. 500 steps of energy minimization using the Powell algorithm followed simulated annealing. For comparison 200 structures each were calculated with and without the introduction of RDCs. After refinement, the structures were evaluated for convergence. Acceptance criteria were low overall energies and no significant NOE (>0.2 Å) or dihedral (>5°) violations. The twenty lowest-energy structures out of 200 calculated were visualized and analyzed using MOLMOL.(29)

Conformational analysis, the wormsearch for comparison of structures, and motif search were performed with the AMIGOS algorithms (http://pylelab.org/software/index.html).(15,16)

4.4.3 Electrostatic surface potential calculation of d3'-EBS1* and d3'- EBS1*·IBS1*

The electrostatic surface potential of d3'-EBS1* and d3'-EBS1*·IBS1* was calculated with the program QNIFFT(80) and visualized with PYMOL (W. L. DeLano, 2002, http://www.pymol.org). The electrostatic potential of nucleic acids was calculated by means of the non-linear Poisson-Boltzmann (NLPB) equation. A surface potential representation elucidates areas of unusual negative or positive potential, which is an indication of potential molecular interactions, e.g. between RNA and metal ions.(80)

4.4.4 Structure calculation of d3'-TL

NOE distances were estimated from the integrated peak volumes obtained from the 2D [1H,1H]-NOESY spectrum that was acquired at 303 K with a mixing time of 250 ms. Distances were calibrated using the CALIBA macro in DYANA.(397) The NOEs were grouped into four categories, corresponding to strong (1.8 – 3 Å), medium (1.8 – 4.5 Å), weak (3.0 – 6.0 Å), and very weak (4.0 – 7.0 Å) (Appendix 28). NOEs obtained from [1H,1H]-NOESY crosspeaks in 90% H2O/10% D2O at 278 K were qualitatively assigned as strong, medium,

weak, or very weak (Appendix 28).

Based on 1D 31P NMR spectra, the torsion angles α and ζ were set to exclude the trans- range (except for G1, which was left unconstrained, as well as C22, where only α was constrained) (Appendix 29).(121) Based on TOCSY experiments with a 45 ms mixing time, nucleotides with strong H1'-H2' and H1'-H3' crosspeaks were restrained to S-type range (δ = 145 ± 20°) (A11), those with absent H1'-H2' crosspeaks to N-type range (δ = 85 ± 20°), and nucleotides with intermediate crosspeak intensities (G1, G10, A12, A13, and C22) were left unconstrained.(34) The other backbone torsional angles (β, γ, ε) were set to standard A-form values in the helical region of the structure (G2-U9, G14-C21). Based on the intranucleotide H1' to aromatic NOEs of a 60 ms NOESY spectrum the torsion angle χ was restrained to anti (–160 ± 20°) for all residues. Additional H-bond restraints were added for base pairs whose existence was proven by 1H-1H crosspeaks across the helix in the imino region and by HN- crosscorrelations from the 2D JNN HNN-COSY spectrum.(118)

200 initial structures were calculated with CNS 1.2 from an extended structure with random initial velocities using NOE distance, dihedral and H-bond restraints.(119) The structures were subsequently refined in XPLOR-NIH.(120) After refinement, the structures were evaluated for convergence. Acceptance criteria were low overall energies and no

Materials and Methods 169

significant NOE (>0.2 Å) or dihedral (>5°) violations. The twenty lowest-energy structures out of 200 calculated were visualized and analyzed using MOLMOL.(29)

4.4.5 Structure calculation of EBS1*·IBS1*

NOE distances were estimated from the integrated peak volumes obtained from the 2D [1H,1H]-NOESY spectrum that was acquired at 288 K with a mixing time of 250 ms. Distances were calibrated using the CALIBA macro in DYANA.(397) The NOEs were grouped into four categories, corresponding to strong (1.8 – 3.0 Å), medium (1.8 – 4.5 Å), weak (3.0 – 6.0 Å), and very weak (4.0 – 7.0 Å) (Appendix 33). NOEs obtained from [1H,1H]-NOESY crosspeaks in 90% H2O/10% D2O at 278 K were qualitatively assigned as strong, medium,

weak, or very weak (Appendix 33).

Based on 1D 31P NMR spectra, the torsion angles α and ζ were set to exclude the trans- range (Appendix 34).(121) Sugar pucker restraints were included based on TOCSY experiments with 45 ms mixing time.(34) For EBS1*·IBS1* all nucleotides were constrained to N-type (δ = 85 ± 20°) due to missing H1'-H2' and H1'-H3' crosspeaks, except for G19 which was left unconstrained. The other backbone torsional angles (β, γ, ε) were set to standard A- form values. Based on the intranucleotide H1' to aromatic NOEs of a 60 ms NOESY spectrum the torsion angle χ was restrained to anti (–160 ± 20°) for all residues. Additional H-bond restraints were added for base pairs whose existence was proven by 1H-1H crosspeaks across the helix in the imino region.

200 initial structures were calculated with CNS 1.2 from an extended structure with random initial velocities using NOE distance, dihedral and H-bond restraints.(119) The structures were subsequently refined in XPLOR-NIH.(120)

After refinement, the structures were evaluated for convergence. Acceptance criteria were low overall energies and no significant NOE (>0.2 Å) or dihedral (>5°) violations. The twenty lowest-energy structures out of 200 calculated were visualized and analyzed using MOLMOL.(29)