Solution structure of tRNAVal from refinement of homology model against residual dipolar coupling and SAXS data

Abstract

A procedure is presented for refinement of a homology model of E. coli tRNA(Val), originally based on the X-ray structure of yeast tRNA(Phe), using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-H(N) RDCs measured with Pf1 phage alignment, and 20 imino N-H(N) RDCs obtained from magnetic field dependent alignment of tRNA(Val). The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA(Phe) by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q (free) = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA(Phe), in agreement with previous NMR-based tRNA(Val) models.

Figure 1

800 MHz ¹H-¹⁵N shift correlation of tRNA^Val. (A) Part of the imino region of the superimposed 2D ¹⁵N-¹H TROSY-HSQC (blue) and regular HSQC (red) correlation spectra of tRNA^Val recorded at 800 MHz, 28 °C, showing correlations for the imino groups of G nucleotides. (B) Expanded cross peak for the TROSY imino correlation of U64, which forms a wobble base pair with G50 and experiences extensive homonuclear ¹H-¹H RDC with adjacent protons. ¹⁵N-¹H/¹H-¹H dipolar cross correlated relaxation results in asymmetry of the unresolved ¹H-¹H multiplet.

Figure 2

Flow diagram of the two-stage process to generate the refined tRNA^Val structure.

Figure 3

SAXS data for tRNA^Val. (A) Desmeared experimental scattering data extrapolated to zero concentration, and their fit to the starting (stage 1; χ ∼ 2.8) and refined (stage 2; χ ∼ 0.9) tRNA^Phe-based models. The inset shows the effect of the structure factor on the raw line-smeared data as a function of concentration. (B) Pairwise distance distributions, P(r), obtained from the Fourier transforms of the zero-concentration-extrapolated data, and the scattering data simulated from the final stage 2 model (red).

Figure 4

Agreement between experimental RDCs and tRNA^Val models. RDC^Pf1 (A), or RDC^MSA (B) vs stage 1 model, generated without SAXS or RDC input data. (C) RDC^Pf1 vs refined (stage 2) model; (D) RDC^MSA vs refined model. In all cases, filled symbols represent RDCs included when carrying out the SVD fit between the experimental data and the structure; open symbols are (A,B) jack-knifed RDC values, predicted from the alignment tensor when that particular RDC was not included in the SVD fit, and (C) cross-validated RDC values, predicted for the refined model calculated without that particular RDC.

Figure 5

Backbone structures of tRNA^Val obtained by refinement of a tRNA^Phe-based homology model. The regularized starting model (stage 1) is shown in blue; the model refined against Pf1 and MSA RDCs in green, and the model that includes both the RDC and SAXS data in red. The models are superimposed by best-fitting nucleotides 1-7 and 66-72. The absence of restraints for nucleotides 73-76 results in their disorder during the refinement simulated annealing protocol when using only RDC data, whereas they adopt a more compact shape when SAXS data are active.