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CCP4BB  June 2012

CCP4BB June 2012

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Subject:

Re: Do my SAXS data agree with the crystal structure?

From:

David Briggs <[log in to unmask]>

Reply-To:

David Briggs <[log in to unmask]>

Date:

Sun, 17 Jun 2012 12:01:25 +0100

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (218 lines)

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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