Global Molecular Structure and Interfaces:  Refining an RNA:RNA Complex Structure Using Solution X-ray Scattering Data

Abstract

We demonstrate in this report that solution X-ray scattering data can be utilized to precisely define the global structure and, therefore, implicitly the interfaces of an RNA:RNA complex. Defining the interfaces among components and the global structure of multicomponent systems is one of the essential problems in understanding biological interactions on a molecular level. However, identifying molecular interfaces is not an easy task for solution NMR spectroscopists. To resolve this problem, a widely used protocol is to prepare various sophisticated but labor-intensive and sometimes costly isotope-labeled samples and apply NMR isotope-filter experiments. 1-4 Moreover, global structures are often underdetermined, due to a general lack of experimentally measured NMRderived restraints that define the overall dimensionalities and shapes of biomacromolecules and complexes in solution, even if residual dipolar coupling has been utilized to provide global orientation restraints. 5 This lack is particularly severe in the structural determination of RNA molecules or complexes, where the proton spin density is much lower than that in protein counterparts, the structures generally tend to be elongated, and there are few options in selective-labeling sample preparation schemes. Furthermore, isotope filter/edited nuclear Overhauser effect (NOE) experiments are in general rather insensitive. Even when there are observable NOEs, assigning them is often challenging and time-consuming.Small-angle X-ray or neutron scattering (SAXS and SANS) data contain information about the overall shape and dimensionality of biomacromolecules in solution 6-8 and have recently been utilized directly to refine protein solution structures in combination with NMR restraints in order to achieve accurate global orders of singlechain multidomain proteins. 9,10 The utilization of SAXS data to define the global structure and consequently identify the interfaces of complexes of an RNA complex has not been reported. We report here a method that utilizes SAXS data to define the global structure and consequently to identify the interfaces of an RNA complex without intermolecular NOE distance restraints and to refine the global shape of the RNA complex. We demonstrate the utility of the method using a 30 kDa homodimeric tetraloop-receptor RNA complex, which is a commonly occurring RNA tertiary structural motif involved in helical packing. 11 The structure of the complex has been determined using heteronuclear solution NMR spectroscopy. 12,13 In the previous determination, the relative position and the orientation between the two subunits were restrained using 36 × 2 intermolecular NOE distance and hydrogen bond restraints together with 9 × 2 imino residual dipolar couplings (RDCs).For a given set of RDCs that are measured in one alignment medium, there are four satisfying discrete orientations for a subunit. In the case of a homodimer with a C 2 -axial symmetry, the orientations of the two subunits are related to each other within four possible choices. 14-16...

Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase

“…5B), EcRapA-TtCore, and EcRapA⌬N-TtCore (Fig. 6B) are also in good agreement with those calculated from experimental data as judged on the basis of previous studies (36,37). Furthermore, we show here that SAXS can be used to complete large, multisubunit structures with missing components.…”

The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes

“…Recent success with combining NMR and SAXS/WAXS data for refining structural ensembles [32,[45][46][47] has shown that inclusion of global restraints is functionally complementary and successful for NMR structure solution, and our work shows that pump-probe X-ray scattering measurements have decisive value for time-resolved structure determination when used in combination with additional experimental restraints from DEER and NMR. b Ramachandran analysis was conducted on the calculated average structure from the ensemble using Molprobity (http://molprobity.biochem.duke.edu/).…”

Reverse Watson-Crick purine-purine base pairs — the Sharp-turn motif and other structural consequences in functional RNAs

“…It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/098723 doi: bioRxiv preprint first posted online Jan. 6, 2017; In the GAAA tetraloop receptor dimers (Table S19), the A:A W:W Trans pair is a part of A:A-U triple that mediates binding of the hairpin GAAA loop of one monomer with the receptor internal loop of the other ( Figure 11F) [79][80][81] . In HIV dimerization initiation site (Context 1 of Table S20) the dimerization of two monomers takes place via a kissing loop interaction 82 .…”