Technical Highlight - October 2009
Short description: Can small angle X-ray scattering bridge the growing gap between structural information and genomic and proteomic advances?
Even with the high-throughput methods of the Protein Structure Initiative and other structural genomics consortia, thousands of proteins have not yielded suitable crystals for structural biology or are too large for NMR spectroscopy studies.
X-ray crystallography has produced the vast majority of structural genomics structures, with NMR proving to be a useful complementary approach. But both these methods have limitations. Small-angle X-ray scattering (SAXS), which provides structural information from X-ray scattering in solution, might provide useful information when these other two methods fail.
According to John Tainer from The Scripps Research Institute in San Diego and his colleagues, SAXS has the potential to be the highest-throughput structural genomics method. In a recent Nature Methods paper 1 , they achieved an 82% success rate using proteins from Pyrococcus furiosus and a selection of proteins from the PSI JCSG. This compares with a roughly 20% success rate for the crystallization samples that were used (and a more success rate of between 3% and 15% for X-ray crystallographic structures).
SAXS has several benefits: its measurements are performed in solution, sample preparation is simple and useful information can be obtained for most proteins. This approach provides solution structural information at a resolution that can give functional insights, although with a resolution of about 15 Å for homogeneous, monodisperse proteins in solution, is it lower than that for X-ray crystallography or NMR.
Tainer's team developed a high-throughput pipeline that largely automates SAXS-based structural analyses and uses the SIBYLS beamline at the Advanced Light Source. Using this set-up, a scattering dataset for each of 96 samples was recorded in 4 hours. Twenty proteins per week can be fully analyzed; thus, more than a thousand macromolecules can be analyzed each year.
To communicate their results and to help with the development of SAXS algorithms they created the website BIOISIS for biologically integrated structures in solution, which they hope will be a helpful resource and database for SAXS structures.
SAXS's ability to examine complexes and conformations in multiple physiological solutions and with various ligands provides much-needed functional insights. In addition, it is likely to enhance crystallization trials by providing feedback on the aggregation state and unstructured regions of a protein. A high-throughput SAXS pipeline is likely to be a useful addition to the structural genomics armory.
G. L. Hura et al. Robust, high-throughput solution structural analyses by small X-ray scattering (SAXS).
Nat Meth. 6, 606-612 (2009). doi:10.1038/nmeth.1353