Small angle X-ray scattering as a complementary tool for high-throughput structural studies

Abstract

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples. Small angle X-ray scattering (SAXS) is a solution technique that provides data on the size and multiple conformations of a sample, and can be used to reconstruct a low-resolution molecular envelope of a macromolecule. In this study, SAXS has been used in a high-throughput manner on a subset of 28 proteins, where structural information is available from crystallographic and/or NMR techniques. These crystallographic and NMR structures were used to validate the accuracy of molecular envelopes reconstructed from SAXS data on a statistical level, to compare and highlight complementary structural information that SAXS provides, and to leverage biological information derived by crystallographers and spectroscopists from their structures. All the ab initio molecular envelopes calculated from the SAXS data agree well with the available structural information. SAXS is a powerful albeit low-resolution technique that can provide additional structural information in a high-throughput and complementary manner to improve the functional interpretation of high-resolution structures.

Figure 1

The observed SAXS data and structural fit to the observed data (continuous line) for samples with crystallographic structure. The ab initio SAXS derived envelopes overlaid with crystallographic structure are also shown illustrating the agreement between the techniques. The samples are numbered as in Tables 1 and 2. Samples 4 and 11 contained a mixture of oligomers and are shown below.

Figure 2

The observed SAXS data and structural fit to the observed data (continuous line) for samples with crystallographic structure and multiple constructs. Ab initio SAXS-derived envelopes are overlaid with crystallographic structure. Sample 17 contained a mixture shown below. The figures are shown to the same approximate scale as those in Figure 1 and the remaining structural representations in Figures 3 and 4.

Figure 3

The observed SAXS data and structural fit to the observed data (continuous line) for samples with NMR structures. The ab initio SAXS-derived envelopes are overlaid with the NMR structures to show the agreement between the data. The figures are shown to approximate scale as in the other structural illustrations and illustrate multiple conformations determined from the NMR data.

Figure 4

Ab initio SAXS-derived envelopes overlaid with NMR and crystallographic structure to show the agreement between the different structural methods. The figures are shown to approximate scale and illustrate multiple conformations determined from the NMR data.

Figure 5

Structures of oligomers based on analysis of the SAXS data and known monomer structure. The ab initio SAXS-derived envelopes are shown assuming a monodisperse solution.