It might also be useful to go through the recent paper by Jacques et al Acta cryst D68:620-6 (2012) towards the standardization of saxs data analysis, diagnostics and reporting.
Best regards,
Savvas
On 17-jun.-2012, at 13:01, David Briggs <[log in to unmask]> 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:
> [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
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