[RASMB] SAXS versus Sed. Vel.

[Kushol Gupta]

Dyche - 

I'm assuming you're taking shape reconstructions and back-calculating S values using hydropro or the like?  I'd advise a lot of caution in expecting that type of precision in the comparison you're making.  What you describe is actually sounds quite good, given you're talking about experiments performed in very different conditions at very different concentrations.  

What is the normalized spatial discrepancy of your shape reconstructions via what method?  What do the Kratky and porod-debye plots looks like?  SAXS is very sensitive to flexibility and lack of compactness, which in turn can exaggerate the molecular volume of the particles determined.  Just because one crystal structure fits a SAXS profile does not mean other structures do not fit the profile as well.  If you have a flexible system, shape reconstructions are generally quite misleading.

Very simply: what are the S values calculated for your crystal structures (assuming a complete inventory)?

Kushol


Kushol Gupta, Ph.D.
Research Associate - Van Duyne Group
Department of Biochemistry and Biophysics
Perelman School of Medicine at The University of Pennsylvania
kgupta at upenn.edu / 215.573.7260 / 267.259.0082 / www.stwing.upenn.edu/~kgupta
-----Original Message-----
From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Mullins, Dyche
Sent: Wednesday, June 17, 2015 3:01 PM
To: rasmb at list.rasmb.org
Subject: [RASMB] SAXS versus Sed. Vel.


Dear All,

We have been looking at nucleotide-dependent conformational changes in several proteins, using both sedimentation velocity and small-angle X-ray scattering (SAXS). Interestingly, we get different results for the magnitude of the nucleotide-induced conformational change and, systematically, the AUC measurement suggests a larger change than the SAXS measurement. For one protein, for example, SAXS predicts sedimentation coefficients of 2.99 and 3.07 for nucleotide-free and nucleotide-bound forms (and for what it’s worth, these values match calculations based on crystal structures of the apo and ADP forms of the protein). By AUC, however, we measure values of 3.00 and 3.15. For the other protein the situations is almost exactly the same: AUC reports a change in S value almost twice that computed from SAXS. 

I suppose pressure might cause additional conformational changes. What else are we missing? 

Thank you,
Dyche Mullins
UCSF
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