[ccp4bb] Free R with doubled cell edge
Dear BB, Thanks for all of your helpful and useful comments Whilst there was some agreement that simulated annealing should be enough to solve any problems, we have taken perhaps the cautious approach. We have produced a freeR set in the bigger unit cell using reindex as suggested by Eleanor: This is easy to do Reindex 2h,k,l then the cell will double; the FreeR will stay with the reflection, and you can use those FreeRs to append to your new data in the scala/truncate GUI. All the unset ones (2h+1,k,l) will be given new FreeRs and the old ones transferred. Eleanor Mostly for the reasons as outlines by Ethan: One problem with shaking things up as you describe is that if the original model was refined against higher resolution data than your new data set, you will probably never get back to the same model quality as you started with (see the ongoing discussion in another thread). And if the new data set is higher resolution, then you face the same problem in reverse. If you want to take your eventual new, higher resolution model back into the old cell you want subsequent refinement to have unbiased Rfree on that end also. Ethan as we now have higher resolution data (than we had for the small unit cell before) for both the small and doubled unit cell - the highest resolution data is for the doubled unit cell with 4 chains, but we would like to refine in the smaller cell as well at some point for completeness. As pointed out, there is a straight translation in the larger unit cell (doubled along a), h(odd) 0 0 reflections are weak, and Phenix Xtriage identifies the translation readily. However, Phaser has correctly positioned 4 chains and we are happily refining in Refmac, so I think that should not be a problem. Many thanks to all who commented on the BB and off line. Cheers Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ People who know nothing about cheeses reel away from Camembert, Roquefort, and Stilton because the plebeian proboscis is not equipped to differentiate between the sordid and the sublime. Harvey Day (1971) Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two-fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a0 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high temp simulated annealing and hope that any bias is gone. I'm not sure that I am comfortable with the word hope here... But, if the consensus of opinion of the wise folk at the BB is that this will pass muster at the point where the charming and delightful referees are commenting on the extremely high impact (obviously :-) manuscript, then I will quote you all! I await your wise words. Free R. Again. Sorry. Cheers Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/
Re: [ccp4bb] Free R with doubled cell edge
This iseasy to do Reindex 2h,k,l then the cell will double; the FreeR will stay with the reflection, and you can use those FreeRs to append to your new data in the scala/truncate GUI. All the unset ones (2h+1,k,l) willbe given new FreeRs and the old ones transferred. Eleanor in On 10/30/2010 06:08 PM, Ethan Merritt wrote: On Saturday, October 30, 2010, Boaz Shaanan wrote: Hi, I'm not sure why you want to carry the free R reflections from the small cell to the new cell. If it's the model bias vis-a vis the reflections participating the refinement that you want to get rid of you can take another route, I think. 1) Select R-free set for the new cell (paying attention to the new NCS); 2) Take the model you obtained after phaser (4 chains) and shake it to death (either in CNS by annealing or in the ccp4 shake routine or the USF equivalent). By then, your model should have got rid of the bias and you can start refinement. Gurus, have I cut any corner by suggesting this route? I think it is better to do exactly what was requested - carry forward the old Rfree to the new data set. Since the a axis is double, the Rfree of smallcell [h k l] is transferred to bigcell [2h k l] One problem with shaking things up as you describe is that if the original model was refined against higher resolution data than your new data set, you will probably never get back to the same model quality as you started with (see the ongoing discussion in another thread). And if the new data set is higher resolution, then you face the same problem in reverse. If you want to take your eventual new, higher resolution model back into the old cell you want subsequent refinement to have unbiased Rfree on that end also. Ethan Would there be any objection from referees (...)? Good luck, Boaz - Original Message - From: Kay Diederichskay.diederi...@uni-konstanz.de Date: Saturday, October 30, 2010 14:23 Subject: Re: [ccp4bb] Free R with doubled cell edge To: CCP4BB@JISCMAIL.AC.UK Hi Ed, in the new cell (long a axis), the reflections H K L are related by H=2*h K=k L=l to those of the old (short a) cell. I would expect that the R-factor of those H K L reflections with even H from the new crystal form is low (at least at low resolution) against the h k l reflections of the old crystal form. (I'd also expect that they are stronger than the odd-H reflections.) You can obtain the R-free flag for these reflections by creating a file with h k l R-free-flag from your old dataset, multiplying all h by 2 (it should be possible to do this with the CCP4 program reindex, using reindex HKL h+h, k, l as the only input line), and using that for the new data. This procedure gives you R-free flags for half of the reflections of your new dataset (those with even H). Those reflections with odd H are of course new; they are not (directly) related to any reflections of the old crystal form. You may want to randomly assign R-free flags to them; there is (I think) a task in ccp4i which takes care of partially missing R-free flags. HTH, Kay Am 20:59, schrieb Thomas Edwards: Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two- fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a0 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion
Re: [ccp4bb] Free R with doubled cell edge
Hi, I'm not sure why you want to carry the free R reflections from the small cell to the new cell. If it's the model bias vis-a vis the reflections participating the refinement that you want to get rid of you can take another route, I think. 1) Select R-free set for the new cell (paying attention to the new NCS); 2) Take the model you obtained after phaser (4 chains) and shake it to death (either in CNS by annealing or in the ccp4 shake routine or the USF equivalent). By then, your model should have got rid of the bias and you can start refinement. Gurus, have I cut any corner by suggesting this route? Would there be any objection from referees (...)? Good luck, Boaz - Original Message - From: Kay Diederichs kay.diederi...@uni-konstanz.de Date: Saturday, October 30, 2010 14:23 Subject: Re: [ccp4bb] Free R with doubled cell edge To: CCP4BB@JISCMAIL.AC.UK Hi Ed, in the new cell (long a axis), the reflections H K L are related by H=2*h K=k L=l to those of the old (short a) cell. I would expect that the R-factor of those H K L reflections with even H from the new crystal form is low (at least at low resolution) against the h k l reflections of the old crystal form. (I'd also expect that they are stronger than the odd-H reflections.) You can obtain the R-free flag for these reflections by creating a file with h k l R-free-flag from your old dataset, multiplying all h by 2 (it should be possible to do this with the CCP4 program reindex, using reindex HKL h+h, k, l as the only input line), and using that for the new data. This procedure gives you R-free flags for half of the reflections of your new dataset (those with even H). Those reflections with odd H are of course new; they are not (directly) related to any reflections of the old crystal form. You may want to randomly assign R-free flags to them; there is (I think) a task in ccp4i which takes care of partially missing R-free flags. HTH, Kay Am 20:59, schrieb Thomas Edwards: Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two- fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a0 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high temp simulated annealing and hope that any bias is gone. I'm not sure that I am comfortable with the word hope here... But, if the consensus of opinion of the wise folk at the BB is that this will pass muster at the point where the charming and delightful referees are commenting on the extremely high impact (obviously :-) manuscript, then I will quote you all! I await your wise words. Free R. Again. Sorry. Cheers Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- A new scientific truth does not triumph by convincing opponents and making them see the light, but rather because its opponents eventually die, and a new generation grows up that is familiar with it. ~Max Planck Boaz Shaanan, Ph.D
Re: [ccp4bb] Free R with doubled cell edge
On Saturday, October 30, 2010, Boaz Shaanan wrote: Hi, I'm not sure why you want to carry the free R reflections from the small cell to the new cell. If it's the model bias vis-a vis the reflections participating the refinement that you want to get rid of you can take another route, I think. 1) Select R-free set for the new cell (paying attention to the new NCS); 2) Take the model you obtained after phaser (4 chains) and shake it to death (either in CNS by annealing or in the ccp4 shake routine or the USF equivalent). By then, your model should have got rid of the bias and you can start refinement. Gurus, have I cut any corner by suggesting this route? I think it is better to do exactly what was requested - carry forward the old Rfree to the new data set. Since the a axis is double, the Rfree of smallcell [h k l] is transferred to bigcell [2h k l] One problem with shaking things up as you describe is that if the original model was refined against higher resolution data than your new data set, you will probably never get back to the same model quality as you started with (see the ongoing discussion in another thread). And if the new data set is higher resolution, then you face the same problem in reverse. If you want to take your eventual new, higher resolution model back into the old cell you want subsequent refinement to have unbiased Rfree on that end also. Ethan Would there be any objection from referees (...)? Good luck, Boaz - Original Message - From: Kay Diederichs kay.diederi...@uni-konstanz.de Date: Saturday, October 30, 2010 14:23 Subject: Re: [ccp4bb] Free R with doubled cell edge To: CCP4BB@JISCMAIL.AC.UK Hi Ed, in the new cell (long a axis), the reflections H K L are related by H=2*h K=k L=l to those of the old (short a) cell. I would expect that the R-factor of those H K L reflections with even H from the new crystal form is low (at least at low resolution) against the h k l reflections of the old crystal form. (I'd also expect that they are stronger than the odd-H reflections.) You can obtain the R-free flag for these reflections by creating a file with h k l R-free-flag from your old dataset, multiplying all h by 2 (it should be possible to do this with the CCP4 program reindex, using reindex HKL h+h, k, l as the only input line), and using that for the new data. This procedure gives you R-free flags for half of the reflections of your new dataset (those with even H). Those reflections with odd H are of course new; they are not (directly) related to any reflections of the old crystal form. You may want to randomly assign R-free flags to them; there is (I think) a task in ccp4i which takes care of partially missing R-free flags. HTH, Kay Am 20:59, schrieb Thomas Edwards: Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two- fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a0 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high
[ccp4bb] Free R with doubled cell edge
Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two-fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a=30 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high temp simulated annealing and hope that any bias is gone. I'm not sure that I am comfortable with the word hope here... But, if the consensus of opinion of the wise folk at the BB is that this will pass muster at the point where the charming and delightful referees are commenting on the extremely high impact (obviously :-) manuscript, then I will quote you all! I await your wise words. Free R. Again. Sorry. Cheers Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- A new scientific truth does not triumph by convincing opponents and making them see the light, but rather because its opponents eventually die, and a new generation grows up that is familiar with it. ~Max Planck
