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Re: [ccp4bb] Do my SAXS data agree with the crystal structure?
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: Do my SAXS data agree with the crystal structure?
From: Kushol Gupta kushol {- dot -} gupta {- at -} GMAIL {- dot -} COM
Date: 2012-06-16		Next message:
Subject: Re: Do my SAXS data agree with the crystal structure?
From: David Briggs drdavidcbriggs {- at -} GMAIL {- dot -} COM
Date: 2012-06-17


Subject: Re: Do my SAXS data agree with the crystal structure?
From: Xun Lu xluncsu {- at -} GMAIL {- dot -} COM
Date: 2012-06-17

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 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 ****
>
> kgupta@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
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