Re: [ccp4bb] Se oxidation
Dear all, one comment on the subject of white lines and anomalous phasing power - one has to be extremely careful to jump to conclusions about the effect that a chemical treatment of the heavy atom (say oxydation/reduction of Se) has on the shape of the edge and the associated anomalous signal: there are crystals where the intrinsic anisotropy of the dispersive and anomalous scattering combines with the geometry of the heavy-atom arrangement and the symmetry of the lattice to give a very strong dependency of the edge (and therefore the signal) on the orientation of the sample in the polarized beam. In other words you may observe a wonderful white line but to check if this improved edge comes from oxydising/reducing the sample that went into the crystal you would need to check it by reorienting the crystal in a number of orientations. And of course one would want to do the same on a crystal comign from the protein before the treatment. And I would not be surprised if in some cases the phenomenon would make the difference between succeeding in determining the structure or not - even when non chemical treatment is involved - just a series of crystals of the same kind but mounted and measured in crucially different orientations. See Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments. Schiltz M, Bricogne G. Acta Crystallogr D Biol Crystallogr. 2008 Jul;D64(Pt 7):711-29. Epub 2008 Jun 18. and references cited therein. Regards Pietro -- Pietro Roversi EP Abraham Fellow in Biochemistry - Lincoln College - Oxford Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385
Re: [ccp4bb] Se oxidation
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 _ Get 5 GB of storage with Windows Live Hotmail. Sign up today. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_11 2008 E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11720 http://www.pctools.com/en/spyware-doctor-antivirus/ -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
[ccp4bb] Se oxidation
Dear AllI 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).ThanksDr. R.DepetrisWeill Cornell Medical College _ Get 5 GB of storage with Windows Live Hotmail. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] Se oxidation
Hi, I wouldn't worry about Se oxidation. In principle having a mix of oxidized/reduced seleniums is unfavorable, as you'll have less signal at the edge (broadening). However, all-oxidized Se apparently makes things better (sharper and more intense peak; I forgot the reference, i think it may have been an Acta Cryst. D paper). We never bother with adding EDTA and/or reducing agents and haven't had problems determining structures by SAD. You should be fine. Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of aka akaka Sent: Mon 2/9/2009 1:27 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 Get 5 GB of storage with Windows Live Hotmail. Sign up today. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] Se oxidation
Hi, I wouldn't bother either. I once phased a structure based on about 160 oxidized Se in the AU (Hothorn et al., JBC, 2006). Just make sure that most of them are oxidized and do a proper absorption scan. best Michael aka akaka wrote: 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 Get 5 GB of storage with Windows Live Hotmail. Sign up today. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] Se oxidation
Hi,I believe it is not important as long as you run a proper scan of the crystal. Both forms will allow proper phasing. This is very well described in a paper from Thomazeau et al. here is the reference MAD on threonine synthase: the phasing power of oxidized selenomethionine. Acta Crystallogr D Biol Crystallogr.javascript:AL_get(this,%20'jour',%20'Acta%20Crystallogr%20D%20Biol%20Crystallogr.'); 2001 Sep;57(Pt 9):1337-40. Epub 2001 Aug 23. Cheers, Pascal Egea, PhD Post Doctoral Researcher UCSF Department of Biochemistry and Biophysics Stroud laboratory On Mon, Feb 9, 2009 at 10:27 AM, aka akaka druida...@hotmail.com wrote: 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 -- Get 5 GB of storage with Windows Live Hotmail. Sign up today.http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
