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Biological Small-Angle X-Ray Solution Scattering (BioSAXS) at MacCHESS

A Need-to-Know Quick Start Guide

 

What is BioSAXS (Biological Small Angle X-Ray Solution Scattering)?

The basic idea behind small angle X-ray solution scattering is simple: shine a beam of X-rays on a droplet of protein solution and measure how much the rays get deflected. Most X-rays travel right through matter without changing direction, but approximately one out of every million gets deflected by a small angle! The very faint “small angle" scattering pattern on an X-ray detector looks a lot like a sunset with the undeflected beam being the like the sun: there is a bright glow nearest the beam that fades to dark at higher angles. To protect our sensitive detector, we block out the direct beam with a small metal rectangle called a beamstop. With modern algorithms, the simple pattern of light to dark on the X-ray detector can yield a surprising amount of valuable structural information about biomolecules. In fact, BioSAXS is becoming an indispensable tool in biomedical science. Not only can researchers tell the mass and size of a biomolecule in solution, but they can reconstruct its basic shape, tell if it is rigid or flexible, and even figure out how multiple molecules fit together to form complex molecular machines. Increasingly, advances in medical research depend upon gaining a clear understanding of how biomolecules function and interact within the living cell. BioSAXS is one of the few techniques that can yield structural information on how biomolecules behave under conditions very similar to the living cell.





 

What parameters can BioSAXS determine?

  • radius of gyration (typically 2% accuracy)
  • molecular weight (typically 10% accuracy)
  • maximum intra-particle distance
  • low-resolution particle shape
  • degree of folding, denaturation, or disorder

What are people able to do with BioSAXS data?

  • determining physiological oligomeric state
  • validating proposed models of complexes
  • building complexes from monomers or known fragments
  • studying protein-protein interaction under different solution conditions
  • modeling missing loops and domains
  • refining homology models
  • categorizing discrete folded and unfolded states
  • finding volume fractions in mixtures

Will BioSAXS work on my samples?

In crystallography, poor crystals and overlapping spots are a frequent cause of failure. BioSAXS can have problems too. Just because it does not require crystals does not guarantee success. In crystallography, it is easier to detect bad data: you can't index or integrate the diffraction spots. With BioSAXS, however, you can process bad data with very few indications that anything is wrong. It is therefore very important to understand how to recognize bad data and to diagnose the possible causes.

Potential problems

  • aggregation (most common)
  • molecule too large (beamline can't reach low enough q)
  • sample is a mixture (common with complexes)1
  • sample too dilute
  • radiation damage
  • denaturation (rare)
  • buffer mismatch
  • contrast problems (weak signal due to buffer composition)
  • heterogeneous sample (protein-DNA-lipid complexes)2

1Generally, solutions must be pure and monodisperse for standard SAXS analysis to work. If you are hoping to see a conformational change when you add a component like a small ligand or other binding partner to the protein sample, be careful to maintain exactly matching buffer. This may mean changing buffer again using a centrifugal concentrator, dialysis, or a SEC run. If conversion of your protein to the new state is incomplete, you may need to re-purify. Mixtures can be treated with BioSAXS, but this is an advanced topic and additional information and multiple experiments may be required to understand the data.

2Mixed complexes of protein, DNA, and/or lipids can cause problems with certain SAXS calculations.

Methods and Acknowledgment

Acerbo, A. S., Cook, M. J., & Gillilan, R. E. (2015). Upgrade of MacCHESS facility for X-ray scattering of biological macromolecules in solution. Journal of Synchrotron Radiation, 22(1), 180-186. doi: doi:10.1107/S1600577514020360

Skou, S., Gillilan, R. E., & Ando, N. (2014). Synchrotron-based small-angle X-ray scattering of proteins in solution. Nature Protocols, 9(7), 1727-1739.

More information:

Jacques, D. A. and J. Trewhella (2010). Small-angle scattering for structural biology — Expanding the frontier while avoiding the pitfalls. Protein Science 19(4): 642-657

Putnam, C. D., M. Hammel, et al. (2007). X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Quarterly Reviews of Biophysics, 40(3): 191-285.

Mertens, H. D. T. and D. I. Svergun (2010). Structural characterization of proteins and complexes using small-angle X-ray solution scattering. Journal of Structural Biology, 172(1): 128-141.

Svergun, D. I. and M. H. J. Koch (2003). Small-angle scattering studies of biological macromolecules in solution. Reports on Progress in Physics, 66(10): 1735-1782.

Feigin, L. A. and D. I. Svergun (1987). Structure Analysis by Small-Angle X-ray and Neutron Scattering, Plenum Press/Springer.

Glatter, O. and Kratky (1982). Small-angle X-ray Scattering. London, Academic Press.

Guinier, A. and G. Fourne (1955). Small-Angle Scattering of X-Rays. New York, John Wiley and Sons.

meetings.chess.cornell.edu/ ACABioSAS/index.html: 2013 ACA BioSAS Training Workshop webpages. Contains tutorials, extensive lecture notes, software links with installation instructions, online tools, and much more.

bl1231.als.lbl.gov/saxs_protocols/saxs_data_collection_evaluatio.php: SIBYLS beamline (ALS)

bioisis.net/tutorial: A useful introductory tutorial in BioSAXS can be found at BIOISIS (ALS)

www.saxier.org/forum/index.php: The Small Angle X-ray Scattering Initiative for Europe (Saxier) also hosts a forum where you can ask questions about BioSAXS processing

http://www.oumedicine.com/docs/default-source/peds-hem-onc-workfiles/small-angle-scattering-links.html?sfvrsn=0: Biomolecular Solution Scattering Links (OU Medicine)

sourceforge.net/projects/bioxtasraw/: "BioXTAS RAW" the processing software we use at MacCHES

www.embl-hamburg.de/ExternalInfo/Research/Sax/software.html: Biological Small Angle Scattering Group (EMBL Hamburg)

situs.biomachina.org/tutorial_saxs.html#visual: Biomachina

www.bioisis.net/tutorial/9: SCATTER developed by Ivan Rodic and Rob Rambo. Estimates molecular weight without knowing concentration, evaluates flexibility, and contains many other important tools

www.synchrotron.org.au/index.php/aussyncbeamlines/saxswaxs/software-saxswaxs: scatterBrain, the SAXS data reduction program developed by the Australian Synchrotron