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. 2010 Mar;16(3):638-46.
doi: 10.1261/rna.1946310. Epub 2010 Jan 27.

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Robert P Rambo  1 , John A Tainer
Affiliations

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Robert P Rambo et al. RNA. 2010 Mar.

Abstract

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA(val), and yeast tRNA(phe) that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.

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Figures

FIGURE 1.
FIGURE 1.
Size exclusion chromatographic (SEC) analysis of SAM-I and P4-P6 domain. Gel-filtration elution profiles for folded RNAs. Vertical axis is the mAu at 280 nm. (A) SAM-I RNA before purification (broken green line) and after SAXS of the purified RNA (red line); (B) P4-P6 domain (solid green line). Arrows mark regions of heterogeneity.
FIGURE 2.
FIGURE 2.
SAXS analyses of refolded SAM-I and P4-P6 domain RNA. (A) Experimental SAXS curves of SAM-plus (red) and SAM-free (black) overlaid with the theoretical scattering curve (orange) calculated from X-ray crystal structure (PDB: 2GIS). The experimental curves were placed on a relative scale in PRIMUS, and the theoretical curve was manually adjusted to illustrate the overall disagreement with the experimental curves. The arrow marks a region of disagreement between the two scattering curves. (B) Kratky plots of the SAM-plus (red) and SAM-free (black) scattering data. (C) Crystallographic and ab initio SAXS models of SAM-I. The surface model (orange) is the ab initio model calculated using the SAM-plus data and represents an average of eight independent particle reconstructions using the program DAMMIF. The SAM-plus data were transformed using a dmax of 118 Å. The X-ray crystal structure (PDB: 2GIS) of SAM-I (orange) is shown as reference with its corresponding dmax. (D) Experimental SAXS curve of the P4-P6 domain (black circles) with the theoretical SAXS profile (orange) calculated from the X-ray crystal structure (PDB: 1L8V). The data were scaled to a common low-angle scattering intensity for purposes of an overlay using PRIMUS. (E) Kratky plot of the P4-P6 domain SAXS data. (F) X-ray crystal structure (orange ribbon or solvent-exposed surface) superimposed with the surface ab initio model (gray surface or wire mesh) calculated from the P4-P6 domain SAXS data, an average of eight independent DAMMIN/F runs using data transformed with a dmax of 178 Å. Crystallographic models were aligned with SUPCOMB20.
FIGURE 3.
FIGURE 3.
SAXS data and models from SEC purified refolded SAM-I and P4-P6 domain. Experimental SAXS profiles of purified SAM-plus (A) and P4-P6 domain (B). In both cases, the orange curve represents the theoretical SAXS profile calculated from X-ray crystal structures of SAM-I (PDB: 2GIS) and P4-P6 domain (PDB: 1L8V). DAMMIN/F ab initio models in two orientations calculated from the SEC purified SAXS data SAM-I RNA (C) and P4-P6 domain (D). Orange backbone models represent the X-ray crystal structures for SAM-I and P4-P6 domain superposed with the program SUPCOMB.
FIGURE 4.
FIGURE 4.
SEC profile and SAXS curves of the 30S subunit, BMV TLS, lysine riboswitch, and tRNAphe. (A) Elution profile of studied RNAs. The signal for the 30S subunit (red) was measured using a refractive index detector and scaled for the figure. Each elution profile was shifted for clarification by 0.3, 0.6, 0.9, and 1.2 mL for BMV TLS (BMV) (green), lysine riboswitch (Lys) (cyan), tRNAVal (Val) (gray), and tRNAphe (Phe) (orange), respectively. SAXS profiles for 30S subunit (B), BMV TLS RNA (C), lysine riboswitch (D) in the presence (cyan) and absence (gray) of lysine, and tRNAphe (orange) and tRNAval (gray) (E). Data for tRNAval were collected with a larger beamstop mask truncating the data at lower scattering angles.
FIGURE 5.
FIGURE 5.
ab initio models of the 30S subunit, BMV TLS, lysine riboswitch, and tRNAphe. (A) S. solfataricus 30S ribosomal subunit; (B) BMV TLS RNA; (C) bound lysine riboswitch; (D) tRNAphe. All ab initio models were created with DAMMIF and averaged from eight independent runs with DAMAVER using SAXS data collected from. SEC purified samples. Two contoured surfaces are used for models where no prior PDB entry is available. For A and B, the two surfaces represented the model at relative contours of 0.5 (wireframe) and 1 (solid). For C and D, the bead models (white) are aligned with the respective crystal structures (orange).
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