Re: [ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps
Thanks to all who replied to my message. The most logical explanation to the observed phenomenon is likely the following. During crystallization, Sr ions from the crystallization solution kick out a fraction of the Mg(OH)6 ions from the binding site in question. The cavity of the binding site is big and the Sr ion binds at a slightly different position compared to the Mg(OH)6 ion. The BDF map shows the shifted position of the Sr ion, whereas the 2Fo-Fc map shows the position of the Mg(OH)6 ion. This is possible because the occupancy of the Sr ion at that site is low - the height of the Sr peak in the BDF map is less than 15% of the maximal peak height - and the contribution of the Sr ion to the 2Fo-Fc map is negligibly small. The “normal” (2Fo-Fc) scattering of the Sr ion is essentially completely masked by the Mg(OH)6 ion scattering because the two overlap (they are 0.47 A apart) and the latter has a 5-6 times higher occupancy. Eleanor’s and Phoebe Rice’s comments were extremely useful in solving this puzzle (at least for me - we will see if the referees agree that this is a solution). This particular comment by Phoebe was really insightful considering she did not see the actual data but only read my somewhat incomplete description: “Could it be a mix of species - partly Sr and partly some non-anomalous-scattering entity that sits in a slightly shifted position?” CCP4BB is an incredible resource full of really clever people! Thanks to all again! Petr On Apr 24, 2014, at 11:39, Petr Leiman petr.lei...@epfl.chmailto:petr.lei...@epfl.ch wrote: Dear All, We are looking for an explanation for a very strange observation. Problem: We have two fully independent data sets (two different crystals), in which the Bijvoet Difference Fourier map peak of one particular metal site is shifted by 0.47 A from its position in the 2Fo-Fc map. Relevant information: The resolution of both data sets is 1.5-1.6 A. The 2Fo-Fc and BDP maps are calculated using the same phases. The metal ion is water hydrated and all the details are crystal clear in both 2Fo-Fc maps. The crystals are grown in the presence of Sr and the data sets are collected at the Sr K-edge. There are many other Sr sites and all strong peaks in the BDF map overlap with 2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but it is well above the noise level of the BDF map. Additional information: The weird site is actually fully buried inside an internal cavity (it is surrounded by protein atoms from all sides), but a Sr atom is able to diffuse into this cavity somehow. All other Sr sites are on the surface of the protein. Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc map peak are welcome! Thank you very much, Petr -- Petr Leiman EPFL BSP 415 CH-1015 Lausanne Switzerland Office: +41 21 69 30 441 Mobile: +41 79 538 7647 Fax: +41 21 69 30 422
[ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps
Dear All, We are looking for an explanation for a very strange observation. Problem: We have two fully independent data sets (two different crystals), in which the Bijvoet Difference Fourier map peak of one particular metal site is shifted by 0.47 A from its position in the 2Fo-Fc map. Relevant information: The resolution of both data sets is 1.5-1.6 A. The 2Fo-Fc and BDP maps are calculated using the same phases. The metal ion is water hydrated and all the details are crystal clear in both 2Fo-Fc maps. The crystals are grown in the presence of Sr and the data sets are collected at the Sr K-edge. There are many other Sr sites and all strong peaks in the BDF map overlap with 2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but it is well above the noise level of the BDF map. Additional information: The weird site is actually fully buried inside an internal cavity (it is surrounded by protein atoms from all sides), but a Sr atom is able to diffuse into this cavity somehow. All other Sr sites are on the surface of the protein. Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc map peak are welcome! Thank you very much, Petr -- Petr Leiman EPFL BSP 415 CH-1015 Lausanne Switzerland Office: +41 21 69 30 441 Mobile: +41 79 538 7647 Fax: +41 21 69 30 422
Re: [ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps
A technecality - the Dano only contains information about anomalous scatterers and should therefore show these more precisely. The 2Fo-Fc map will tend to show anything which is included in the phasing at the position given. For safety you can set all the atom occupancies in the vicinity of the site to 0.00 and then use phases calculated from the edited structure to check again. ... This sort of thing sometimes happens with the Se of MSE, and the explanation there is that the MSE is in more than one conformation.. Eleanor On 24 April 2014 10:39, Petr Leiman petr.lei...@epfl.ch wrote: Dear All, We are looking for an explanation for a very strange observation. *Problem*: We have *two fully independent data sets* (two different crystals), in which the Bijvoet Difference Fourier map peak of one particular metal site is shifted by 0.47 A from its position in the 2Fo-Fc map. *Relevant information*: The resolution of both data sets is 1.5-1.6 A. The 2Fo-Fc and BDP maps are calculated using the same phases. The metal ion is water hydrated and all the details are crystal clear in both 2Fo-Fc maps. The crystals are grown in the presence of Sr and the data sets are collected at the Sr K-edge. There are many other Sr sites and all strong peaks in the BDF map overlap with 2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but it is well above the noise level of the BDF map. *Additional information*: The weird site is actually fully buried inside an internal cavity (it is surrounded by protein atoms from all sides), but a Sr atom is able to diffuse into this cavity somehow. All other Sr sites are on the surface of the protein. Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc map peak are welcome! Thank you very much, Petr -- Petr Leiman EPFL BSP 415 CH-1015 Lausanne Switzerland Office: +41 21 69 30 441 Mobile: +41 79 538 7647 Fax: +41 21 69 30 422
Re: [ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps
Hi Petr, A few thoughts: 1) The Bijvoet difference Fourier is an approximation to the true imaginary part of the electron density, and because of this shifts in the positions of peaks in this map relative to the 2Fo-Fc map might be expected. This is described in Hendrickson et al. Acta Cryst A43 121-125. 2) It might be possible that the responsible anomalous scatterer is not Sr but something else that is lighter (although perhaps not likely given the wavelength the data was collected at). If a heavier atom is incorrectly modeled in place of a lighter one, it can exacerbate the spurious shifts in the BDF peaks. This is discussed in the reference above. I guess the same thing could occur if the site of interest is less than fully occupied but modeled with full occupancy. 3) It might be informative to verify the anomalous scatterer positions with imaginary LLG maps, which can be calculated using Phaser (or SHARP). Philip On Thu, Apr 24, 2014 at 5:39 AM, Petr Leiman petr.lei...@epfl.ch wrote: Dear All, We are looking for an explanation for a very strange observation. *Problem*: We have *two fully independent data sets* (two different crystals), in which the Bijvoet Difference Fourier map peak of one particular metal site is shifted by 0.47 A from its position in the 2Fo-Fc map. *Relevant information*: The resolution of both data sets is 1.5-1.6 A. The 2Fo-Fc and BDP maps are calculated using the same phases. The metal ion is water hydrated and all the details are crystal clear in both 2Fo-Fc maps. The crystals are grown in the presence of Sr and the data sets are collected at the Sr K-edge. There are many other Sr sites and all strong peaks in the BDF map overlap with 2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but it is well above the noise level of the BDF map. *Additional information*: The weird site is actually fully buried inside an internal cavity (it is surrounded by protein atoms from all sides), but a Sr atom is able to diffuse into this cavity somehow. All other Sr sites are on the surface of the protein. Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc map peak are welcome! Thank you very much, Petr -- Petr Leiman EPFL BSP 415 CH-1015 Lausanne Switzerland Office: +41 21 69 30 441 Mobile: +41 79 538 7647 Fax: +41 21 69 30 422
Re: [ccp4bb] Systematically shifted peak in 2Fo-Fc and BDF maps
Dear Petr Leiman : It could be the result of series termination in anomolous map.(not sure) The error in the position results in a shifted position in the 2Fo-Fc map. As a check it is worthwhile to see an average of the two positions (three coordinates) results in a feature consistent with Bijvoet Difference and 2Fo-Fc maps. But I do not know how this error affects just one site and not others. Best Srinivasan Raghunathan On Thu, 24 Apr 2014, Petr Leiman wrote: Dear All, We are looking for an explanation for a very strange observation. Problem: We have two fully independent data sets (two different crystals), in which the Bijvoet Difference Fourier map peak of one particular metal site is shifted by 0.47 A from its position in the 2Fo-Fc map. Relevant information: The resolution of both data sets is 1.5-1.6 A. The 2Fo-Fc and BDP maps are calculated using the same phases. The metal ion is water hydrated and all the details are crystal clear in both 2Fo-Fc maps. The crystals are grown in the presence of Sr and the data sets are collected at the Sr K-edge. There are many other Sr sites and all strong peaks in the BDF map overlap with 2Fo-Fc map peaks perfectly. The Sr site in question is not the strongest, but it is well above the noise level of the BDF map. Additional information: The weird site is actually fully buried inside an internal cavity (it is surrounded by protein atoms from all sides), but a Sr atom is able to diffuse into this cavity somehow. All other Sr sites are on the surface of the protein. Any thoughts about why a non-noise BDF map peak would not overlap with a 2Fo-Fc map peak are welcome! Thank you very much, Petr -- Petr Leiman EPFL BSP 415 CH-1015 Lausanne Switzerland Office: +41 21 69 30 441 Mobile: +41 79 538 7647 Fax: +41 21 69 30 422
