If you main point is dimer vs monomer, Guinier plots is in fact all you would need, in my opinion according to what I have read so far...
A. On 17 Jun 2012, at 13:01, David Briggs wrote: > 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: > david.c.bri...@manchester.ac.uk > ============================ > Webs : http://flavors.me/xtaldave > Twitter: @xtaldave > Skype: DocDCB > ============================ > > > On 17 June 2012 06:11, Xun Lu <xlun...@gmail.com> 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 <kushol.gu...@gmail.com> >> 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 >>> >>> kgu...@mail.med.upenn.edu >>> >>> 215-573-7260 / 267-259-0082 >>> >>> >>> >>> >>> >>> -----Original Message----- >>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xun >>> Lu >>> Sent: Saturday, June 16, 2012 2:29 PM >>> To: CCP4BB@JISCMAIL.AC.UK >>> 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
