Nucleic acid structure characterization by small angle X-ray scattering (SAXS).
ABSTRACT: Small angle X-ray scattering (SAXS) is a powerful method for investigating macromolecular structure in solution. SAXS data provide information about the size and shape of a molecule with a resolution of ?2 to 3 nm. SAXS is particularly useful for the investigation of nucleic acids, which scatter X-rays strongly due to the electron-rich phosphate backbone. Therefore, SAXS has become an increasingly popular method for modeling nucleic acid structures, an endeavor made tractable by the highly regular helical nature of nucleic acid secondary structures. Recently, SAXS was used in combination with NMR to filter and refine all-atom models of a U2/U6 small nuclear RNA complex. In this unit, general protocols for sample preparation, data acquisition, and data analysis and processing are given. Additionally, examples of correctly and incorrectly processed SAXS data and expected results are provided.
Project description:NMR and SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) are highly complementary approaches for the analysis of RNA structure in solution. Here we describe an efficient NMR-SAXS/WAXS approach for structural investigation of multi-helical RNAs. We illustrate this approach by determining the overall fold of a 92-nt 3-helix junction from the U4/U6 di-snRNA. The U4/U6 di-snRNA is conserved in eukaryotes and is part of the U4/U6.U5 tri-snRNP, a large ribonucleoprotein complex that comprises a major subunit of the assembled spliceosome. Helical orientations can be determined by X-ray scattering data alone, but the addition of NMR RDC (residual dipolar coupling) restraints improves the structure models. RDCs were measured in two different external alignment media and also by magnetic susceptibility anisotropy. The resulting alignment tensors are collinear, which is a previously noted problem for nucleic acids. Including WAXS data in the calculations produces models with significantly better fits to the scattering data. In solution, the U4/U6 di-snRNA forms a 3-helix junction with a planar Y-shaped structure and has no detectable tertiary interactions. Single-molecule Förster resonance energy transfer data support the observed topology. A comparison with the recently determined cryo-electron microscopy structure of the U4/U6.U5 tri-snRNP illustrates how proteins scaffold the RNA and dramatically alter the geometry of the U4/U6 3-helix junction.
Project description:The U2/U6 snRNA complex is a conserved and essential component of the active spliceosome that interacts with the pre-mRNA substrate and essential protein splicing factors to promote splicing catalysis. Here we have elucidated the solution structure of a 111-nucleotide U2/U6 complex using an approach that integrates SAXS, NMR, and molecular modeling. The U2/U6 structure contains a three-helix junction that forms an extended "Y" shape. The U6 internal stem-loop (ISL) forms a continuous stack with U2/U6 Helices Ib, Ia, and III. The coaxial stacking of Helix Ib on the U6 ISL is a configuration that is similar to the Domain V structure in group II introns. Interestingly, essential features of the complex--including the U80 metal binding site, AGC triad, and pre-mRNA recognition sites--localize to one face of the molecule. This observation suggests that the U2/U6 structure is well-suited for orienting substrate and cofactors during splicing catalysis.
Project description:A generalized method, termed Fast-SAXS-pro, for computing small angle x-ray scattering (SAXS) profiles of proteins, nucleic acids, and their complexes is presented. First, effective coarse-grained structure factors of DNA nucleotides are derived using a simplified two-particle-per-nucleotide representation. Second, SAXS data of a 18-bp double-stranded DNA are measured and used for the calibration of the scattering contribution from excess electron density in the DNA solvation layer. Additional test on a 25-bp DNA duplex validates this SAXS computational method and suggests that DNA has a different contribution from its hydration surface to the total scattering compared to RNA and protein. To account for such a difference, a sigmoidal function is implemented for the treatment of non-uniform electron density across the surface of a protein/nucleic-acid complex. This treatment allows differential scattering from the solvation layer surrounding protein/nucleic-acid complexes. Finally, the applications of this Fast-SAXS-pro method are demonstrated for protein/DNA and protein/RNA complexes.
Project description:The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing.
Project description:Small angle x-ray scattering (SAXS) is a versatile and widely used technique for obtaining low-resolution structures of macromolecules and complexes. SAXS experiments measure molecules in solution, without the need for labeling or crystallization. However, radiation damage currently limits the application of SAXS to molecules that can be produced in microgram quantities; for typical proteins, 10-20 μL of solution at 1 mg/mL is required to accumulate adequate signal before irreversible x-ray damage is observed. Here, we show that cryocooled proteins and nucleic acids can withstand doses at least two orders of magnitude larger than room temperature samples. We demonstrate accurate T = 100 K particle envelope reconstructions from sample volumes as small as 15 nL, a factor of 1000 smaller than in current practice. Cryo-SAXS will thus enable structure determination of difficult-to-express proteins and biologically important, highly radiation-sensitive proteins including light-activated switches and metalloenzymes.
Project description:The removal of introns from pre-messenger RNA is mediated by the spliceosome, a large complex composed of many proteins and five small nuclear RNAs (snRNAs). Of the snRNAs, the U6 and U2 snRNAs are the most conserved in sequence, as they interact extensively with each other and also with the intron, in several base pairings that are necessary for splicing. We have isolated and sequenced the genes encoding both U6 and U2 snRNAs from the intracellularly parasitic microsporidian Nosema locustae . Both genes are expressed. Both RNAs can be folded into secondary structures typical of other known U6 and U2 snRNAs. In addition, the N.locustae U6 and U2 snRNAs have the potential to base pair in the functional intermolecular interactions that have been characterized by extensive analyses in yeast and mammalian systems. These results indicate that the N.locustae U6 and U2 snRNAs may be functional components of an active spliceosome, even though introns have not yet been found in microsporidian genes.
Project description:Exploiting the experimental information from small-angle X-ray solution scattering (SAXS) in conjunction with structure prediction algorithms can be advantageous in the case of ribonucleic acids (RNA), where global restraints on the 3D fold are often lacking. Traditional usage of SAXS data often starts by attempting to reconstruct the molecular shape ab initio, which is subsequently used to assess the quality of a model. Here, an alternative strategy is explored whereby the models from a very large decoy set are directly sorted according to their fit to the SAXS data. For rapid computation of SAXS patterns, the method developed here makes use of a coarse-grained representation of RNA. It also accounts for the explicit treatment of the contribution to the scattering of water molecules and ions surrounding the RNA. The method, called Fast-SAXS-RNA, is first calibrated using a tRNA (tRNA-val) and then tested on the P4-P6 fragment of group I intron (P4-P6). Fast-SAXS-RNA is then used as a filter for decoy models generated by the MC-Fold and MC-Sym pipeline, a suite of RNA 3D all-atom structure algorithms that encode and exploit RNA 3D architectural principles. The ability of Fast-SAXS-RNA to discriminate native folds is tested against three widely used RNA molecules in molecular modeling benchmarks: the tRNA, the P4-P6, and a synthetic hairpin suspected to assemble into a homodimer. For each molecule, a large pool of decoys are generated, scored, and ranked using Fast-SAXS-RNA. The method is able to identify low-rmsd models among top ranking structures, for both tRNA and P4-P6. For the hairpin, the approach correctly identifies the dimeric state as the solution structure over the monomeric state and alternative secondary structures. The method offers a powerful strategy for recognizing native RNA conformations as well as multimeric assemblies and alternative secondary structures, thus enabling high-throughput RNA structure determination using SAXS data.
Project description:Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly.
Project description:Small-angle X-ray scattering is an increasingly popular technique used to detect protein structures and ensembles in solution. However, the refinement of structures and ensembles against SAXS data is often ambiguous due to the low information content of SAXS data, unknown systematic errors, and unknown scattering contributions from the solvent. We offer a solution to such problems by combining Bayesian inference with all-atom molecular dynamics simulations and explicit-solvent SAXS calculations. The Bayesian formulation correctly weights the SAXS data versus prior physical knowledge, it quantifies the precision or ambiguity of fitted structures and ensembles, and it accounts for unknown systematic errors due to poor buffer matching. The method further provides a probabilistic criterion for identifying the number of states required to explain the SAXS data. The method is validated by refining ensembles of a periplasmic binding protein against calculated SAXS curves. Subsequently, we derive the solution ensembles of the eukaryotic chaperone heat shock protein 90 (Hsp90) against experimental SAXS data. We find that the SAXS data of the apo state of Hsp90 is compatible with a single wide-open conformation, whereas the SAXS data of Hsp90 bound to ATP or to an ATP-analogue strongly suggest heterogenous ensembles of a closed and a wide-open state.
Project description:Slt11p is a new splicing factor identified on the basis of synthetic lethality with a mutation in the 5' end of U2 snRNA, a region that is involved in intermolecular U2/U6 helix II interaction. Slt11p is required for spliceosome assembly. Our genetic results suggest that Slt11p is involved in the base-pairing interaction of U2/U6 helix II in vivo. We showed that the recombinant protein binds to RNAs with some degree of structural specificity. Slt11p also anneals RNA and binds to the resulting duplexes, which contain two separated helical regions. These RNA structures are reminiscent of U2/U6 helix II, which is formed concomitantly with U4/U6 stem II, and suggest that Slt11p facilitates the cooperative formation of helix II in association with stem II in the spliceosome. We show that Slt11p and Slu7p, a second-step factor, interact with each other both in vivo and in vitro and that the binding of Slu7p to Slt11p impairs the RNA-binding activity of the latter. These results suggest that the function of Slt11p is regulated by Slu7p in the spliceosome.