NMR-based Structural Characterization of Protein-RNA Complexes

NMR spectra were acquired at 25°C on an 850 MHz Bruker Avance spectrometer, equipped with a triple-resonance (¹⁵N/¹³C/¹H) cryoprobe. The sample volume was either 0.16 or 0.35 mL, in SEC buffer, 5% D₂O/90-95% H₂O. A series of double- and triple-resonance spectra [25 (link),26 (link)] were recorded to obtain sequence-specific resonance assignment. We used the I-PINE assignment tool [27 (link)] implemented in NMRFAM-SPARKY [28 (link)] for initial automatic assignment. ¹H-¹H distance restraints were derived from 3D ¹⁵N/¹H NOESY-HSQC and ¹³C/¹H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.
Structural calculation was carried out in CYANA [29 (link)] using NOESY data in combination with backbone torsion angle restraints, generated from assigned chemical shifts using the program TALOS+ [30 (link)]. First, the combined automated NOE assignment and structure determination protocol (CANDID) was used for automatic NOE cross-peak assignment. Subsequently, five cycles of simulated annealing combined with redundant dihedral angle restraints were used to calculate a set of converged structures with no significant restraint violations (distance and van der Waals violations < 0.5Å and dihedral angle constraint violations < 5°). The 40 structures with the least restraint violations were further refined in explicit solvent using the YASARA software with the YASARA forcefield [18 (link)] and subjected to further analysis using the Protein Structure Validation Software suite (www.nesg.org). The statistics for the resulting structure are summarized in Table 1. The structures, NMR restraints and resonance assignments were deposited in the Protein Data Bank (PDB, accession code: 6YI3) and BMRB (accession code: 34511).
To follow changes in the chemical shifts of a protein upon RNA binding, we calculated chemical shift perturbations (CSPs). The CSP of each assigned resonance in the 2D ¹⁵N/¹H HSQC spectra of the protein in the free state was calculated as the geometrical distance in ppm to the peak in the 2D ¹⁵N/¹H HSQC spectra acquired under different conditions using the formula: $\mathrm{\Delta }\delta =\sqrt{{\mathrm{\Delta }{\delta }_H}^2+({\mathrm{\Delta }{\delta }_N\bullet \alpha )}^2}$ , where α is a weighing factor of 0.2 used to account for differences in the proton and nitrogen spectral widths [31 (link)].