Re: [ccp4bb] Differentiating bound Mn Ca.
Hey David, You can do Mn2+ identification by its anomalous diffraction using the Cu Kalpha radiation. Mn is an anomalous scatterer at Cu Kalpha (1.5418 A), despite being distant from its absorption edge (somewhere around 1.96 A if I remember well). I did this for a double-manganese bound ConA, have a look into THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 19778–19787, 2000 (you may need to use different programs for making your anomalous difference Patterson and Fourier maps these days). Julie. Kay Diederichs wrote: David Briggs wrote: Dear all. I have recently solved a structure in-house, 2.8A, CuKa. I have a metal ion bound very obvious hepta-valent co-ordination, which would suggest either Ca or Mn. Neither was present in the crystallisation setup, but there was some Mg around, which has contaminants of both Ca Mn. At 2.8A, I don't really think I can reliably discriminate between 2.15A 2.36A distances to coordinating atoms (http://tanna.bch.ed.ac.uk/newtargs_06.html http://tanna.bch.ed.ac.uk/newtargs_06.html). The B factors for refined Ca are 18, and Mn 30. The B-factors of coordinating atoms vary from... 18 30 - so no help there. I have a nice clear 6sigma anomalous difference peak, but then, according to http://skuld.bmsc.washington.edu/scatter/ both Ca (f ~1.3) and Mn (f ~2.8) scatter anomalously at that wavelength. The obvious solution is go to a synchrotron and scan around the Mn edge and see what happens, however, whilst waiting for beam time, is there any way I could... oh I don't know, use the peak in my anomalous difference Fourier to figure out what anomalous signal would be required to generate a peak of that size - a sort of back-transform??? Is this do-able, and if so, how would one go about it? Cheers, Dave Dave, f = 1.3 versus 2.8 sounds like quite a difference ... what is the anom peak height of the sulfurs in your structure? The f of sulfur at Cu Ka wavelength is 0.55 . So I'd expect the ratio of peakheights of your unknown metal divided by the average peakheight of sulfurs (of roughly 18-30 A**2 B-factor) to give you an idea of what you have. Of course this is no proof ... Are there any other anom scatterers in your structure? best, Kay -- Julie Bouckaert, PhD[EMAIL PROTECTED] VIB Project leader VIB Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel Tel. 32-2-629-1988 Fax 32-2-6291963 ULTR, Building E 4.18 Vrije Universiteit Brussel Pleinlaan 2 1050 Brussel Belgium
Re: [ccp4bb] Differentiating bound Mn Ca.
Hi David, You can use Sheldrick's Calcium Bond Valence Sum to descriminate between metals (see Muller, P., Kopke, S., and Sheldrick, G. M. (2003) Acta Crystallogr., Sect. D: Biol. Crystallogr. 59, 32-37) even at low resolution. I have had good success with this method combined with estimation of anomalous contribution scaled against the S atoms as suggested by Kay. Have a look at gratuitous self plug Graham, S. C., Bond, C. S., Freeman, H. C., and Guss, J. M. (2005) Biochemistry 44, 13820-13836. /gratuitous self plug for plenty of examples. To do the anomalous difference calculations I just integrated a 2x2x2 A box around the metal atom and around the S atoms (in MAPMAN) then calculated the ratio of anomalous difference... Cheers, Stephen On 4/16/07, David Briggs [EMAIL PROTECTED] wrote: Dear all. I have recently solved a structure in-house, 2.8A, CuKa. I have a metal ion bound very obvious hepta-valent co-ordination, which would suggest either Ca or Mn. Neither was present in the crystallisation setup, but there was some Mg around, which has contaminants of both Ca Mn. At 2.8A, I don't really think I can reliably discriminate between 2.15A 2.36A distances to coordinating atoms (http://tanna.bch.ed.ac.uk/newtargs_06.html ). The B factors for refined Ca are 18, and Mn 30. The B-factors of coordinating atoms vary from... 18 30 - so no help there. I have a nice clear 6sigma anomalous difference peak, but then, according to http://skuld.bmsc.washington.edu/scatter/ both Ca (f ~1.3) and Mn (f ~2.8) scatter anomalously at that wavelength. The obvious solution is go to a synchrotron and scan around the Mn edge and see what happens, however, whilst waiting for beam time, is there any way I could... oh I don't know, use the peak in my anomalous difference Fourier to figure out what anomalous signal would be required to generate a peak of that size - a sort of back-transform??? Is this do-able, and if so, how would one go about it? Cheers, Dave -- --- David Briggs, PhD. Father Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] Differentiating bound Mn Ca.
In cases like this I use the S atoms to calibrate the peak height. Of course it isnt definitive a) it is near the noise level, and b) peak height is very dependent on B factor.. But the ratio might distinguish between an atom with an f of 1.3 or f=2.8 Eleanor David Briggs wrote: Dear all. I have recently solved a structure in-house, 2.8A, CuKa. I have a metal ion bound very obvious hepta-valent co-ordination, which would suggest either Ca or Mn. Neither was present in the crystallisation setup, but there was some Mg around, which has contaminants of both Ca Mn. At 2.8A, I don't really think I can reliably discriminate between 2.15A 2.36A distances to coordinating atoms (http://tanna.bch.ed.ac.uk/newtargs_06.html http://tanna.bch.ed.ac.uk/newtargs_06.html). The B factors for refined Ca are 18, and Mn 30. The B-factors of coordinating atoms vary from... 18 30 - so no help there. I have a nice clear 6sigma anomalous difference peak, but then, according to http://skuld.bmsc.washington.edu/scatter/ both Ca (f ~1.3) and Mn (f ~2.8) scatter anomalously at that wavelength. The obvious solution is go to a synchrotron and scan around the Mn edge and see what happens, however, whilst waiting for beam time, is there any way I could... oh I don't know, use the peak in my anomalous difference Fourier to figure out what anomalous signal would be required to generate a peak of that size - a sort of back-transform??? Is this do-able, and if so, how would one go about it? Cheers, Dave -- --- David Briggs, PhD. Father Crystallographer www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams
Re: [ccp4bb] Differentiating bound Mn Ca.
Although the peak height of S atoms can be used as an internal yardstick, one has to worry about differences in occupancy and possibly hetergeneous sites (i.e. Ca, Mn and Mg) which can confuse the interpretation of the results. On Mon, 16 Apr 2007, Eleanor Dodson wrote: In cases like this I use the S atoms to calibrate the peak height. Of course it isnt definitive a) it is near the noise level, and b) peak height is very dependent on B factor.. But the ratio might distinguish between an atom with an f of 1.3 or f=2.8 Eleanor David Briggs wrote: Dear all. I have recently solved a structure in-house, 2.8A, CuKa. I have a metal ion bound very obvious hepta-valent co-ordination, which would suggest either Ca or Mn. Neither was present in the crystallisation setup, but there was some Mg around, which has contaminants of both Ca Mn. At 2.8A, I don't really think I can reliably discriminate between 2.15A 2.36A distances to coordinating atoms (http://tanna.bch.ed.ac.uk/newtargs_06.html http://tanna.bch.ed.ac.uk/newtargs_06.html). The B factors for refined Ca are 18, and Mn 30. The B-factors of coordinating atoms vary from... 18 30 - so no help there. I have a nice clear 6sigma anomalous difference peak, but then, according to http://skuld.bmsc.washington.edu/scatter/ both Ca (f ~1.3) and Mn (f ~2.8) scatter anomalously at that wavelength. The obvious solution is go to a synchrotron and scan around the Mn edge and see what happens, however, whilst waiting for beam time, is there any way I could... oh I don't know, use the peak in my anomalous difference Fourier to figure out what anomalous signal would be required to generate a peak of that size - a sort of back-transform??? Is this do-able, and if so, how would one go about it? Cheers, Dave -- --- David Briggs, PhD. Father Crystallographer www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams
