KRATKY ANALYSIS ... by Robert P. Rambo, Ph.D.

The "unfolded-ness" or "random coil" likeness of your biological macromolecule can be qualitatively assessed by means of a Kratky plot. The plot uses your merged data and can be created using any plotting or spreadsheet program but we will demonstrate it using ScÅtter. Conveniently, ScÅtter IV creates Kratky plot for each dataset in Analysis panel as a small plot in upper right.

Courtesy of Putnam, C.D., Hammel, M., Hura, G.L., and Tainer, J.A. Q Rev Biophys. 2007 40(3):191-285

The Kratky interpretation originates from Debye's scattering formulation of a Gaussian coil. Debye's equation shows that within a limited range of data, the scattering intensities for macromolecules behaving as Gaussian-like coils will plateau in a q2 x I(q) vs q plot. To begin, load some data and click on the Kratky button (third column from right in Analysis panel, Figure 1). Here, we have loaded two datasets, glucose isomerase (blue) and the SAM-I riboswitch (green).

Figure 1:

The Kratky button transforms the data and plots (Figure 2). In this case, I previously scaled the two datasets together so that they are close together in the Kratky plot. Unscaled, peak heights provide no meaningful interpretation. What is important is how the transformed SAXS datasets converge to baseline. As you can see, glucose isomerase does not plateau whereas SAM-I does. These are extreme cases that compare a compact versus a flexible particle. SAM-I is an RNA riboswitch that requires Mg2+ and s-adensyl-methionine to fold into a discrete compact structure. This SAXS data of SAM-I is in the absence of the metabolite. In many cases, the presence of a plateau will be followed by a slow descent to baseline for partially flexible particles. More evidence can be gained through examining the particle volume and Porod exponent. In some cases, poor buffer subtraction can lead artificially to elevated baselines at high q.

Also, if you see in Figure 1, glucose isomerase and SAM-I have similar Rg values though SAM-I is 4 times smaller than glucose isomerase. The Rg comparison strongly supports the notion that the mass distribution of SAM-I is wide and dispersed over a large space leading to a relatively large Rg value (as we would expect for an unfolding of the RNA). A semi-quantitative analysis can be performed using the "Normalized Kratky" plot.

Figure 2: