Dear Savvas,
Since you mention the case of TolC explicitly, I should add to what
Pietro Roversi has already written in his message in relation to anisotropy
effects. Ben Luisi's group kindly let us have their data in the late 20th
century, so that we could use the then new and exciting log-likelihood
gradient maps in SHARP to take a look at the Se atoms, expecting to see some
features around them that would have indicated at least partial oxidation.
To our surprise, there was no hint of any such extra density.
Later, in papers that paved the way towards the work referred to in
Pietro's message, it was pointed out that the anisotropy of anomalous
scattering can sometimes result in white lines being visible or not in the
Se fluorescence spectra, depending on the orientation of the crystal. The
effets can be very strong, and an "unlucky" orientation for some crystals
with a polar axis can be seriously detrimental. See:
X-ray absorption, refraction and resonant scattering tensors in selenated
protein crystals: implications for data collection strategies in
macromolecular crystallography (2005). G. Bricogne, S. C. Capelli, G.
Evans, A. Mitschler, P. Pattison, P. Roversi & M. Schiltz. J. Appl.
Cryst. 38, 168-182.
Polarization-dependence of anomalous scattering in brominated DNA and RNA
molecules, and importance of crystal orientation in single- and
multiple-wavelength anomalous diffraction phasing (2007). R. Sanishvili,
C. Besnard, F. Camus, M. Fleurant, P. Pattison, G. Bricogne & M. Schiltz.
J. Appl. Cryst. 40, 552-558.
In section 3.6 of the first paper, a discussion is given of the
possibility that what had been interpreted as an oxidation effect (leading
to the loss of a white line at the Se K-edge) in selenated N-Myristoyl
transferase could plausibly have been an anisotropy (i.e. polarisation
dependence) effect. No such re-examination of the TolC case has so far been
performed, and now would clearly be a good time to do that.
To get back to the initial question: any attempt to interpret in purely
chemical terms (e.g. oxidation) what may modify the appearance of the Se
K-edge should bear in mind that a physical effect - the polarisation
dependence of anomalous scattering - may also be involved in some cases. As
shown in the paper referred to in Pietro's message, this was seen as a
nuisance and was ignored for the past 20 years, but it can be exploited to
extract more phase information from the same datasets; and it is possible to
design experiments specifically so as to maximise the amount of extra phase
information that this will deliver.
With best wishes,
Gerard.
--
On Tue, Feb 10, 2009 at 08:05:49AM +0100, Savvas Savvides wrote:
> I think that SeMet oxidation has been a problem in the past in at least one
> case that I know, that of TolC by Koronakis et al. The same group addressed
> these problems in more detail in a second paper (see below):
>
>
>
> Crystal structure of the bacterial membrane protein TolC central to
> multidrug efflux and protein export.
>
> Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C.
>
> Nature. 2000 Jun 22;405(6789):914-9.
>
>
>
> Oxidation of selenomethionine: some MADness in the method!
>
> Sharff AJ, Koronakis E, Luisi B, Koronakis V.
>
> Acta Crystallogr D Biol Crystallogr. 2000 Jun;56(Pt 6):785-8.
>
>
>
> Best wishes
>
> Savvas
>
>
>
> ----
> Savvas Savvides
> L-ProBE, Unit for Structural Biology
> Ghent University
> K.L. Ledeganckstraat 35
> 9000 Ghent, BELGIUM
> office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
> Email: savvas.savvi...@ugent.be
> http://www.lprobe.ugent.be/xray.html
>
>
>
>
>
>
>
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of aka
> akaka
> Sent: Monday, February 09, 2009 7:27 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Se oxidation
>
>
>
> Dear All
>
>
>
> I would like to know whether oxidation of Se entails any problem for SAD or
> MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents
> in my protein (extracellular and disulphide bonds are important).
>
> Thanks
>
>
>
> Dr. R.Depetris
>
> Weill Cornell Medical College
>
>
>
>
>
>
>
> _____
>
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