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Dear Xun, Regarding your monomer vs dimer, theoretical vs observed crysol plots - yes - they are significantly different. If you focus at the very lowest q part of the curve - the deviation there in your monomer plots indicate that there is a significant size difference between your PX monomer and your SAXS data - the PX dimer is a much better fit at low q. This should be enough to demonstrate to a reviewer that the dimer you see in PX is also present in solution. Other experiments that could support this are SEC-MALLS or perhaps AUC. HTH, Dave ============================ David C. Briggs PhD Father, Structural Biologist and Sceptic ============================ University of Manchester E-mail: [log in to unmask] ============================ Webs : http://flavors.me/xtaldave Twitter: @xtaldave Skype: DocDCB ============================ On 17 June 2012 06:11, Xun Lu <[log in to unmask]> wrote: > Drs.Caldwell, Briggs, and Gupta, > > Thank you very much for the advices. I regret that I didn't show any > figure in the earlier post. Here I've attached a figure showing the data > quality and some fittings. > Data look OK, right? This question may sound silly, but I just want to > make sure. > As I said in the earlier post, I tried Crysol. I used the crystal > structure (dimer+DNA) as the model, and the fitting was OK, right? In fact, > I also tried monomer+DNA as the model (I simply deleted one monomer from the > PDB file). This kind of comparison may be meaningless, but I was just > curious. I am wondering how people judge whether the fit is good or not. > > > Another question, I tried to generate an envelope from SAXS data using > Gasbor and Dammin (people say Dammin is better at protein-DNA complex, > although it still uses the same bead for both DNA and protein?). The > generated envelope was nothing like my crystal structure. As people have > pointed out, protein and DNA scatter differently. SANS is the way to go. > So I should give up on modeling SAXS data? I've almost given up, because > anyways I have the crystal structure, and SAXS is only a small part of this > paper. > > > > Thanks, > > Xun > > > > On Sat, Jun 16, 2012 at 6:36 PM, Kushol Gupta <[log in to unmask]> > wrote: >> >> Two cents - >> >> >> >> A good deal of caution must be exercised when working with composite >> particles such as a protein-DNA complex in SAXS because of the contrast >> problem. Simply, protein and DNA scatter differently in x-rays, with a bias >> towards the DNA component. As a result, experimental Rgs could be slightly >> deflated versus what their true values would be at infinite contrast. Mass >> estimation by I(0) analysis with a protein standard of known mass and >> concentration is not really valid because the contrast terms are different. >> Because the particle is heterogeneous in composition and distribution, shape >> reconstruction from SAXS alone, which assumes homogeneity, can also be >> misleading (although in practice it is still reasonably instructive). It is >> for these reasons that SANS and the contrast variation approach can be >> extremely useful. >> >> >> >> With those caveats, the strategy you describe - comparison of experimental >> and theoretical profiles from an experimental structure using CRYSOL or FoxS >> is definitely the best way to go in the case of a protein-DNA complex with >> SAXS alone. Showing comparisons of the experimental with the calculated >> should make the point. Test other possible models inferred from lattice >> packing to further your point (if applicable). >> >> >> >> Regarding populations of monomer and dimer - >> >> >> >> · it is generally good to constrain your interpretation of >> scattering data with other orthogonal solution measures which demonstrates >> the homogeneity of your complex in comparable experimental conditions, such >> as sedimentation velocity or gel filtration. >> >> >> >> · Have some determination of affinity of the complex in the same >> solution conditions (including temperature!). This will allow you to argue >> that your sample concentrations are well in excess of any monomer-dimer >> association behavior (eg, mixtures!). Scattering of mixtures can undermine >> your ability to accurately assess the structural properties of your complex. >> >> >> >> · Collect a concentration series and extrapolate to infinite >> dilution, if possible, to ensure elimination of the S(q) term from your >> data. Interparticle interactions can be an issue with complexes containing >> DNA if the buffers aren’t quite right. (I’ve seen this a lot) >> >> >> >> Lastly, remember that the scattering profile represents the solution >> average of the particle, not just a single snapshot. Some discrepancies >> like those you note should be expected. >> >> >> >> Hope that helps, >> >> >> >> Kushol >> >> >> >> Kushol Gupta, Ph.D. >> >> Research Associate - Van Duyne Laboratory >> >> HHMI / Perelman School of Medicine >> >> University of Pennsylvania >> >> [log in to unmask] >> >> 215-573-7260 / 267-259-0082 >> >> >> >> >> >> -----Original Message----- >> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Xun >> Lu >> Sent: Saturday, June 16, 2012 2:29 PM >> To: [log in to unmask] >> Subject: [ccp4bb] Do my SAXS data agree with the crystal structure? >> >> >> >> Dear all, >> >> >> >> >> >> I have solved a protein-DNA structure, and I also did SAXS to get >> some ideas of the solution structure. The SAXS data were good, no >> aggregation at all three tested concentrations. I tried to use Crysol to >> see if my crystal structure fits the SAXS. The fitting to the scattering >> profile seems good to me and the Chi2 is 1~1.4. Then I wanted to see how >> the P(r) looked like (wanted to make a figure for my paper:). I calculated >> the theoretical scattering profile of the crystal structure from an online >> server (FOXS). I then run GNOM to make P(r). To my surprise, this >> theoretical P(r) looks a little different from the P(r) of SAXS data. >> There's a very small bump that was peaked at 70A (Dmax is 108A, which seems >> reasonable from the crystal structure). The major peak was at 25A. As >> some people said, P(r) is indeed quite sensitive to subtle differences. >> >> >> >> The protein is a dimer in the crystal, although it can also bind >> DNA as a monomer (much more loosely). The estimated MW from SAXS indicates >> it's a dimer in solution as well. It seems that I got the information I >> wanted from the SAXS experiment, but maybe not. Due to the low resolution >> of SAXS, maybe I can only say that the majority is a dimer?? Would it be >> possible to see the monomer if there's only 10% of them in the solution? >> How to interpret the discrepancy between the P(r) from crystal and the P(r) >> from SAXS? >> >> >> >> >> >> Any comments are welcome! >> >> >> >> >> >> >> >> >> >> Xun >> >> >> >> >> >> Sent from my iPad= > > > > > -- > Department of Molecular and Structural Biochemistry > North Carolina State University