Re: [ccp4bb] Translational pseudosymmetry?
This looks a bit strange.. If you have a hexamer in the asymmetric unit, in P3, then that means all symmetry copies lie in the same plane. To generate the Patterson peak, 2/3,1/3,0 the hexamer must be centred at 1/3,1/3, z (with symmetry equivalents 0,-1/3,z and -1/3,0,z ) I would expect ypu to have a pseudo higher symmetry SG - does pointless make any suggestions? Eleanor Jürgen Bosch wrote: Hi Owen, you should also make the following plot with your data: y-axis relative intensity of off-origin peak versus x-axis resolution cutoff used for calculation (30 Å - 4 Å in 2 Å steps). You can have multiple cases of shifts and I would start with a perfect hexamer first, take some random monomer and apply a perfect sixfold, move it along the axis where it should be in your crystal lattice (things get more complicated if you have a top/down hexamer, so keep it simple). Now if you shift your hexamer to 2/3,1/3,0 your plot should yield a straight line and be independent of resolution. Now start rotating the second hexamer relative to the first clockwise with your sixfold, I would use increments of 3 degrees, which will result in 19 models, then recalculate the off-origin peak heights and see if they match up with your data. I should note here, if your real data does not show a strong drop in peak height of the off-origin peak, then you most likely don't have a slight rotational translation in your second hexamer. One other important thing you should look at is the relative orientation of your sixfold axis, is it truly perfectly aligned with one of the cell axis ? If not fix this in your model, otherwise your calculations will be of academic nature. For this particular case the use of GLRF is more helpful than MOLREP (sorry Garib, but maybe Garib can come up with a solution to zoom into certain peaks like you can do in GLRF). When the tilt is fixed you should be able to figure out the rotational translation in your second hexamer. Enjoy your puzzle, Jürgen P.S. P3 is certain ? Check with pointless or by human brain visual inspection (HBVI) On May 15, 2010, at 11:53 PM, Owen Pornillos wrote: Dear ccp4bb – I have questions with regards to crystal disorder that gives rise to translational pseudosymmetry. We have a rotationally hexameric protein that crystallized in P3, with one hexamer in the asu. The local 6-fold axis of the hexamer is non- crystallographic, and is essentially parallel to the crystallographic 3-fold, which gave rise to translational pseudosymmetry. Intensities for the (h,h+/-3n,l) reflections were on average about 8 times stronger than the weak reflections, and the native patterson gave an off-origin peak about 70-80% of origin (depending on the crystal) at fractional coordinates (2/3,1/3,0). We are hypothesizing that the break in local 6-fold symmetry is caused by small rigid-body displacements of each subunit (as opposed to conformational changes in the protein), and we are trying to estimate the magnitude of the displacements in the crystal. To do this, a perfectly symmetric hexamer with the local 6-fold axis parallel to the crystallographic 3-fold was generated, and then shifts were introduced to the atomic coordinates. The direction of the shift was chosen randomly for each atom, and a single magnitude applied to all atoms, which was then changed incrementally. Structure factors were calculated from these models, and their pattersons were examined. The magnitude of the off-origin peak could be reproduced with an atomic shift of say, 1 Å. Because all of these calculations were made with synthetic structure factors, this is not necessarily a reliable estimate. The questions are, how far off are we, and in what direction (i.e., are these shifts underestimates or overestimates)? Is there a way to obtain a reliable estimate? Thanks in advance, Owen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Translational pseudosymmetry?
Dear Eleanor - That is correct. The pseudo-sg is P6, and the structure has been refined in this sg. The intensity difference between the strong and weak subsets is quite significant that for most data sets, auto- indexing routines will miss the weak spots and pick the pseudo-sg instead. The pseudo-sg is a'=b'=90, c'=56, the true sg is a=b=156, c=56. Note that a = a' * sqrt(3). So, the sg assignment is certain. Owen On May 20, 2010, at 6:33 AM, Eleanor Dodson wrote: This looks a bit strange.. If you have a hexamer in the asymmetric unit, in P3, then that means all symmetry copies lie in the same plane. To generate the Patterson peak, 2/3,1/3,0 the hexamer must be centred at 1/3,1/3, z (with symmetry equivalents 0,-1/3,z and -1/3,0,z ) I would expect ypu to have a pseudo higher symmetry SG - does pointless make any suggestions? Eleanor Jürgen Bosch wrote: Hi Owen, you should also make the following plot with your data: y-axis relative intensity of off-origin peak versus x-axis resolution cutoff used for calculation (30 Å - 4 Å in 2 Å steps). You can have multiple cases of shifts and I would start with a perfect hexamer first, take some random monomer and apply a perfect sixfold, move it along the axis where it should be in your crystal lattice (things get more complicated if you have a top/down hexamer, so keep it simple). Now if you shift your hexamer to 2/3,1/3,0 your plot should yield a straight line and be independent of resolution. Now start rotating the second hexamer relative to the first clockwise with your sixfold, I would use increments of 3 degrees, which will result in 19 models, then recalculate the off- origin peak heights and see if they match up with your data. I should note here, if your real data does not show a strong drop in peak height of the off-origin peak, then you most likely don't have a slight rotational translation in your second hexamer. One other important thing you should look at is the relative orientation of your sixfold axis, is it truly perfectly aligned with one of the cell axis ? If not fix this in your model, otherwise your calculations will be of academic nature. For this particular case the use of GLRF is more helpful than MOLREP (sorry Garib, but maybe Garib can come up with a solution to zoom into certain peaks like you can do in GLRF). When the tilt is fixed you should be able to figure out the rotational translation in your second hexamer. Enjoy your puzzle, Jürgen P.S. P3 is certain ? Check with pointless or by human brain visual inspection (HBVI) On May 15, 2010, at 11:53 PM, Owen Pornillos wrote: Dear ccp4bb – I have questions with regards to crystal disorder that gives rise to translational pseudosymmetry. We have a rotationally hexameric protein that crystallized in P3, with one hexamer in the asu. The local 6-fold axis of the hexamer is non- crystallographic, and is essentially parallel to the crystallographic 3-fold, which gave rise to translational pseudosymmetry. Intensities for the (h,h+/-3n,l) reflections were on average about 8 times stronger than the weak reflections, and the native patterson gave an off-origin peak about 70-80% of origin (depending on the crystal) at fractional coordinates (2/3,1/3,0). We are hypothesizing that the break in local 6-fold symmetry is caused by small rigid-body displacements of each subunit (as opposed to conformational changes in the protein), and we are trying to estimate the magnitude of the displacements in the crystal. To do this, a perfectly symmetric hexamer with the local 6-fold axis parallel to the crystallographic 3-fold was generated, and then shifts were introduced to the atomic coordinates. The direction of the shift was chosen randomly for each atom, and a single magnitude applied to all atoms, which was then changed incrementally. Structure factors were calculated from these models, and their pattersons were examined. The magnitude of the off-origin peak could be reproduced with an atomic shift of say, 1 Å. Because all of these calculations were made with synthetic structure factors, this is not necessarily a reliable estimate. The questions are, how far off are we, and in what direction (i.e., are these shifts underestimates or overestimates)? Is there a way to obtain a reliable estimate? Thanks in advance, Owen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Translational pseudosymmetry?
Hi Owen, you should also make the following plot with your data: y-axis relative intensity of off-origin peak versus x-axis resolution cutoff used for calculation (30 Å - 4 Å in 2 Å steps). You can have multiple cases of shifts and I would start with a perfect hexamer first, take some random monomer and apply a perfect sixfold, move it along the axis where it should be in your crystal lattice (things get more complicated if you have a top/down hexamer, so keep it simple). Now if you shift your hexamer to 2/3,1/3,0 your plot should yield a straight line and be independent of resolution. Now start rotating the second hexamer relative to the first clockwise with your sixfold, I would use increments of 3 degrees, which will result in 19 models, then recalculate the off-origin peak heights and see if they match up with your data. I should note here, if your real data does not show a strong drop in peak height of the off-origin peak, then you most likely don't have a slight rotational translation in your second hexamer. One other important thing you should look at is the relative orientation of your sixfold axis, is it truly perfectly aligned with one of the cell axis ? If not fix this in your model, otherwise your calculations will be of academic nature. For this particular case the use of GLRF is more helpful than MOLREP (sorry Garib, but maybe Garib can come up with a solution to zoom into certain peaks like you can do in GLRF). When the tilt is fixed you should be able to figure out the rotational translation in your second hexamer. Enjoy your puzzle, Jürgen P.S. P3 is certain ? Check with pointless or by human brain visual inspection (HBVI) On May 15, 2010, at 11:53 PM, Owen Pornillos wrote: Dear ccp4bb – I have questions with regards to crystal disorder that gives rise to translational pseudosymmetry. We have a rotationally hexameric protein that crystallized in P3, with one hexamer in the asu. The local 6-fold axis of the hexamer is non- crystallographic, and is essentially parallel to the crystallographic 3-fold, which gave rise to translational pseudosymmetry. Intensities for the (h,h+/-3n,l) reflections were on average about 8 times stronger than the weak reflections, and the native patterson gave an off-origin peak about 70-80% of origin (depending on the crystal) at fractional coordinates (2/3,1/3,0). We are hypothesizing that the break in local 6-fold symmetry is caused by small rigid-body displacements of each subunit (as opposed to conformational changes in the protein), and we are trying to estimate the magnitude of the displacements in the crystal. To do this, a perfectly symmetric hexamer with the local 6-fold axis parallel to the crystallographic 3-fold was generated, and then shifts were introduced to the atomic coordinates. The direction of the shift was chosen randomly for each atom, and a single magnitude applied to all atoms, which was then changed incrementally. Structure factors were calculated from these models, and their pattersons were examined. The magnitude of the off-origin peak could be reproduced with an atomic shift of say, 1 Å. Because all of these calculations were made with synthetic structure factors, this is not necessarily a reliable estimate. The questions are, how far off are we, and in what direction (i.e., are these shifts underestimates or overestimates)? Is there a way to obtain a reliable estimate? Thanks in advance, Owen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
[ccp4bb] Translational pseudosymmetry?
Dear ccp4bb – I have questions with regards to crystal disorder that gives rise to translational pseudosymmetry. We have a rotationally hexameric protein that crystallized in P3, with one hexamer in the asu. The local 6-fold axis of the hexamer is non- crystallographic, and is essentially parallel to the crystallographic 3-fold, which gave rise to translational pseudosymmetry. Intensities for the (h,h+/-3n,l) reflections were on average about 8 times stronger than the weak reflections, and the native patterson gave an off-origin peak about 70-80% of origin (depending on the crystal) at fractional coordinates (2/3,1/3,0). We are hypothesizing that the break in local 6-fold symmetry is caused by small rigid-body displacements of each subunit (as opposed to conformational changes in the protein), and we are trying to estimate the magnitude of the displacements in the crystal. To do this, a perfectly symmetric hexamer with the local 6-fold axis parallel to the crystallographic 3-fold was generated, and then shifts were introduced to the atomic coordinates. The direction of the shift was chosen randomly for each atom, and a single magnitude applied to all atoms, which was then changed incrementally. Structure factors were calculated from these models, and their pattersons were examined. The magnitude of the off-origin peak could be reproduced with an atomic shift of say, 1 Å. Because all of these calculations were made with synthetic structure factors, this is not necessarily a reliable estimate. The questions are, how far off are we, and in what direction (i.e., are these shifts underestimates or overestimates)? Is there a way to obtain a reliable estimate? Thanks in advance, Owen
