Data extrapolation required?

Interactive and automated data processing tools (PRIMUS, GNOM, AUTORG).
Scattering from simple bodies (BODIES), peak analysis (PEAK), data plotting (SASPLOT) etc.
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dapereir
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Joined: 2018.06.14 17:41

Data extrapolation required?

#1 Post by dapereir » 2018.06.14 18:13

Hello,

I am comparing the same protein (2 domains+linker) with point mutations. I measure at batch mode using >6 concentrations collected from SEC performed previously, and protein behaves well (I mean no obvious aggregation and is surely monomeric). I want to verify whether these mutations extend the protein, and whether it affects flexibility (visually checked via Kratky for now, not EOM yet).

QUESTION 1: the wild-type and mutant A are fine, different concentrations do not change Rg or Dmax, MW estimation is also within the error. However if I extrapolate this curve using mutant A, I see changes in Dmax (from average 10.7-11.0 for 6 concentrations down to 8 nm for the extrapolated one, which is quite substantial difference). I am not sure why that happens with the extrapolated curve, since I do not see clear signs of aggregation in the samples used for extrapolation, and buffer subtraction was properly done.

QUESTION 2: Regard mutant B, I see a linear correlation of Rg and Dmax depending on the concentration (higher concentration slightly superestimate these parameters). Thus in this case it seems there is a structural factor affecting the data. This can be minimised from extrapolation using the 7 curves from different concentrations or this data cannot be used? Or any other suggestion how to proceed please?

Since the extrapolated data for Mutant A provides different Dmax then the subtracted curves, I am not sure if I should compare only extrapolate data between the mutants, or if it's fine to compare a subtracted curve for mutant A vs. extrapolated for mutant B.

Below you can find the files for mutant A and B in case you can to have a look.
Thanks in advance.
Attachments
Mutant_B.zip
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Mutant_A.zip
(140.5 KiB) Downloaded 5 times

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