Deriving quantitative dynamics information for proteins and RNAs using ROTDIF with a graphical user interface

Abstract

To facilitate rigorous analysis of molecular motions in proteins, DNA, and RNA, we present a new version of ROTDIF, a program for determining the overall rotational diffusion tensor from single-or multiple-field Nuclear Magnetic Resonance (NMR) relaxation data. We introduce four major features that expand the program’s versatility and usability. The first feature is the ability to analyze, separately or together, 13C and/or 15N relaxation data collected at a single or multiple fields. A significant improvement in the accuracy compared to direct analysis of R2/R1 ratios, especially critical for analysis of 13C relaxation data, is achieved by subtracting high-frequency contributions to relaxation rates. The second new feature is an improved method for computing the rotational diffusion tensor in the presence of biased errors, such as large conformational exchange contributions, that significantly enhances the accuracy of the computation. The third new feature is the integration of the domain alignment and docking module for relaxation-based structure determination of multi-domain systems. Finally, to improve accessibility to all the program features, we introduced a graphical user interface (GUI) that simplifies and speeds up the analysis of the data. Written in Java, the new ROTDIF can run on virtually any computer platform. In addition, the new ROTDIF achieves an order of magnitude speedup over the previous version by implementing a more efficient deterministic minimization algorithm. We not only demonstrate the improvement in accuracy and speed of the new algorithm for synthetic and experimental 13C and 15N relaxation data for several proteins and nucleic acids, but also show that careful analysis required especially for characterizing RNA dynamics allowed us to uncover subtle conformational changes in RNA as a function of temperature that were opaque to previous analysis.

Structural fidelity and NMR relaxation analysis in a prototype RNA hairpin

“…We summarize these data in Table 3 together with estimates from simulation and the hydroNMR program that is often used to estimate diffusion tensors from structure (Garcia De La Torre et al 2000). Recently, some of the NMR relaxation rates used here as reference for EC2 case (Duchardt and Schwalbe 2005;Ferner et al 2008) have been refitted simultaneously to yield similar overall tumbling times, but significantly larger anisotropies (Berlin et al 2013) and no information on the order parameters.…”

“…e (Berlin et al 2013) estimates are from a simultaneous refitting of the data presented in footnotes c and d for dipolar vectors located on the rigid regions of the hairpin.…”

The structure of the SOLE element of oskar mRNA

“…Here, we used the program ROTDIF (Berlin et al 2013) to analyze our 13 C relaxation data (R 1 , R 2 and heteronuclear NOE), measured at a magnetic field of 14.1 T (150.9-MHz carbon frequency), and model both the diffusion tensor and the parameters of internal dynamics. We performed the analysis with two sets of data, comprising either all residues or only the PS stem.…”

“…The relaxation rates R 1 , R 2 , and heteronuclear NOEs were analyzed using ROTDIF (Berlin et al 2013). For τ c > 3 ns the relaxation rates are dominated by the low frequency spectral density components J(0) and J(ω C ).…”

“…This assumption is not correct for the base carbons: The base shielding tensors are approximately axially symmetric (δ 11 ∼δ 33 ), but the unique axis δ 22 is rather perpendicular than collinear to the dipolar interaction tensor (Sitkoff and Case 1998;Stueber and Grant 2002;Hansen and Al-Hashimi 2006). Nonetheless, the program was applied successfully to fit relaxation data of DNA and RNA molecules (Berlin et al 2013). To avoid the selection of complex motional models that may be caused by these systematic errors, we multiplied the errors for the measured relaxation rates by a factor of three, since the selection of local dynamics is based on statistical arguments.…”