SUBTRACTION ... by Robert P. Rambo, Ph.D.

ScÅtter offers a buffer subtraction routine that uses normalized integrated intensities. Normalized implies each I(q) has been corrected for exposure time and beam fluctuations. The normalization is typically performed by the instrument or beamline during data collection.

ScÅtter offers several strategies for performing a buffer subtraction. At B21 (Diamond Light Source), data for a sample is collected as a set of 1 second exposures. Each 1-second exposure is written as a normalized 3 column text file. For a 30-second exposure, we would get 30 frames that comprise the measurement of the sample. Likewise, for the buffer/background, we would expect a similar number of frames. Files are loaded by dropping the appropriate *.dat file in either the "SAMPLES" or "BACKGROUND" panel. The files should be loaded in chronological order from earliest to latest exposure.

Figure 1:

After loading the frames, it is best to assess the stability of the SAXS signal throughout the exposure. Protein samples may aggregate during long exposures (Figure 2, green arrow). Ideally, for a perfect sample, the SAXS curves will perfectly overlay. Radiation damage will be seen as a separation of the SAXS curves in the overlay (green arrow, red curve). Radiation damage will be seen as a change in the similarity plot (orange arrow). The similarity is based off the first curve, here only the first 4 curve are usable in the Sample.

In contrast, the frames corresponding to the buffer show no radiation sensitivity and are ideally similar through the exposure as the similarity plot is relatively flat (cyan arrow).

Figure 2:

To perform the subtraction, select first 4 frames in Samples and all the frames in Buffer. This can be done with the mouse (Figure 3, orange and cyan arrows) by clicking on the left and moving the mouse to the right to the last useable frame. Alternatively, frames can be selected by typing in the start and end indices in the top middle boxes. In this case, the 0 to 4 and 0 to 17 represent selected frames for Sample and Buffer frames respectively.

Figure 3:

Subtraction will be performed by averaging the buffer frames and subtracting the averaged buffer SAS curve from each frame in Sample. To output an average subtracted curve, simple check the box under MERGING.

Unsubtracted SAXS Signal

A SAXS measurement of your macromolecule is really a measurement of two: 1) the buffer(background) scattering and 2) the background plus particle(s) scattering. Consequently, the SAXS profile of the particle, under dilute conditions, is determined as the difference between the scattering of the two samples. Therefore, it is absolutely essential that the buffer environment between the two samples match as closely as possible with the only difference due to the particle(s).

Figure 1:

Figure 1 shows the same buffer measured at two exposures: 300 (left) and 1 (right) seconds. Clearly, the variance of the signal is greatly reduced in the 300 second exposure, particularly in the high q region (q > 0.1). Overlaid in each, is a sample measurement at 1 second exposure. As you can see at q > 0.2, the differences are small if not negligible and it would be difficult to assert that the differences observed at q > 0.2 for both 1 sec (right) exposures is meaningful.

Figure 2:

We can assert that information is present by taking the ratio of the intensities of the sample (particle+background) to the background. Assuming an ideal buffer matching, the presence of signal would be indicated by a ratio greater than 1 (Figure 2). Clearly, in the low q region, a significant peak exists suggesting the presence of a strong signal that decays towards a constant baseline. The peak is an artifact of the instrument and suggests, to the left of the peak, the signal is decaying (likely due to scattering from the beamstop). In the high q region (right panel), a significant fraction of the ratio > 1, suggesting information is present albeit weak. We can use this plot as a means of detecting a SAXS signal that does not require buffer subtraction nor a determined Guinier region. It can be expected that the area under the ratio plot (integral) will scale with concentration and particle mass. Therefore, for a set of SAXS curves collected during an size-exclusion chromatography (SEC) run, a plot of the integral of the ratio for each SAXS measurement will generate a trace of the elution peak.