Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Andrey, Due to our company policy, I cannot view the dropbox image, but the packing you describe is exactly what Edward Barry described and what I expected. Two molecules obey the 2-fold symmetry and one molecule is lying on a 2-fold with two possible superimposed orientations. The question is: are there frequent switches between both conformations such that X-rays diffracted from both conformations are interfering, or are there just larger chunks of crystal with either conformation A or B, behaving like separate crystals? In the first case, one has a crystal packing disorder and, as Eleanor was hinting at, some of the diffraction spots may look funny or smeared, in the second case one has twinning. I do not know the exact method sfcheck uses, but normally to detect twinning, programs do not look at symmetry but look at intensity distributions. E.g. in case of twinning, intensities are averaged and less reflections will have extreme (very high or very low) values and reflections will have more average values. I would expect that if you expand an untwinned P21212 data set to P21 and run the twin test, it still will come out untwinned. So if sfcheck claims the data is twinned, it most likely is and you have used the correct procedure. And in this case you should also process the data in the lower symmetry space group. What wonders me a little is that MR did not give clear solutions in P21 with twinned data. P21 is low symmetry and I would have expected 2 clear solutions, each corresponding to one of the two twin possibilities. Also, if the detwin procedure does what I think it does, it should not perform well if the twin fraction is very close to 0.5 (perfect twinning). As has been pointed out by others, you will have to generate the freeR flags in P21212 and expand them to P21, otherwise your free reflections are linked to working reflections and you will get artificially low free Rfactors. Also, Rfactors and free Rfactors will generally be lower if you use twin refinement. No reflections are discarded during twin refinement, what happens is that both twin orientations are taken into account during refinement (and for the calculation of Rfactors!). Congratulations, it looks like you have solved your problem! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrey Nascimento Sent: Thursday, March 28, 2013 12:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse - even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that solved the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present - P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.ukmailto:eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Appu, I am sorry, I do not have the script file. I ran it basically from default parameters in CCP4 GUI. I just ran the sfcheck for 'check experimental data only' and I got the twin law and twin fraction from the output (post script file). So, I put these information on the detwin and ran it. I will send a .pdf file with the a print screen of ccp4 GUI. I am not a experienced crystallographer, but I hope it helps you. Good luck, Andrey 2013/3/28 Appu kumar appu.kum...@gmail.com Respected sir, I have same problem what you have. I am running the detwin on mtz file but it getting failed. Could you please tell me how you did this. If possible send me your script file. It will be a great help for me. Thank you in advance. Appu On 28 March 2013 05:10, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse – even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that “solved” the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present – P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Herman, I would like to mention one more information, maybe I have forgotten. When a process the data in P21212 and run the sfcheck it do not appear to have twinning (even when a ran phenix Xtriage with older process data in P21212). I will send direct to your e-mail the .pdf file with sfcheck analysis to both space groups. Thank you very much. Andrey 2013/3/28 Andrey Nascimento andreynascime...@gmail.com Dear Appu, I am sorry, I do not have the script file. I ran it basically from default parameters in CCP4 GUI. I just ran the sfcheck for 'check experimental data only' and I got the twin law and twin fraction from the output (post script file). So, I put these information on the detwin and ran it. I will send a .pdf file with the a print screen of ccp4 GUI. I am not a experienced crystallographer, but I hope it helps you. Good luck, Andrey 2013/3/28 Appu kumar appu.kum...@gmail.com Respected sir, I have same problem what you have. I am running the detwin on mtz file but it getting failed. Could you please tell me how you did this. If possible send me your script file. It will be a great help for me. Thank you in advance. Appu On 28 March 2013 05:10, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse – even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that “solved” the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present – P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse – even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that “solved” the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present – P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model building (AutoBuild and ARP/wARP) but they cannot build anything that make some sense or build a random chains without any sense. I do not have an extensive knowledge of crystallography, but I have been thinking about some questions: If the third molecule (the bad one) is lying on the 2-fold symmetry axis on P 2 21 21, and since it does not have an intrinsic 2-fold symmetry axis (like protein molecule), how can I merge the structure factors (or intensities) related by symmetry and expand to lower symmetry afterwards? In this case the molecule lying on the 2-fold symmetry axis will have the structure factors wrongly merged, since the molecule is not symmetric, is it ok? If the third molecule is lying on the 2-fold symmetry axis on P 2 21 21, and only another two molecules can be related by the
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Seems reasonable: P21 with beta=90 is pretty common, and it often ends up twinned as well. Most importantly, your R-factors support the hypothesis. (Only keep in mind that R-factors are lower in twinned refinement - look on Garib's webpage for the presentation where he describes that -- sorry, I don't remember the exact slides.) phx On 27/03/2013 23:40, Andrey Nascimento wrote: Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse – even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that “solved” the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present – P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.uk mailto:eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.com mailto:andreynascime...@gmail.com wrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model building (AutoBuild and ARP/wARP) but they cannot build anything that make some sense or build a random chains without any sense. I do not have an
Re: [ccp4bb] Strange density in solvent channel and high Rfree
First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model building (AutoBuild and ARP/wARP) but they cannot build anything that make some sense or build a random chains without any sense. I do not have an extensive knowledge of crystallography, but I have been thinking about some questions: If the third molecule (the bad one) is lying on the 2-fold symmetry axis on P 2 21 21, and since it does not have an intrinsic 2-fold symmetry axis (like protein molecule), how can I merge the structure factors (or intensities) related by symmetry and expand to lower symmetry afterwards? In this case the molecule lying on the 2-fold symmetry axis will have the structure factors wrongly merged, since the molecule is not symmetric, is it ok? If the third molecule is lying on the 2-fold symmetry axis on P 2 21 21, and only another two molecules can be related by the crystallographic symmetry, is it a case of pseudo-symmetry? But in this case, the third molecule is disordered in the crystal packing (as Zbyszek said), and probably have a long range disorder, because I cannot get a good maps for this third molecule even in P1. (pseudo-symmetry + order/disorder). And a more philosophical question… what is the problem in process data in a lower symmetry? Are there mathematical/statistical problems related that can lead to “false-good” data? I put a new .pdf file (ccp4bb_maps_P21.pdf) with map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps_P21.pdf I am sorry for so many questions and thanks in advance. Cheers, Andrey 2013/3/20 Jrh jrhelliw...@gmail.com Dear Zbyszek, I am concerned that the unmerged data would be bypassed and not preserved in your recommendation. I also find it counter intuitive that the merged data would then be unmerged into a lower symmetry and be better than the unmerged data; there is I imagine some useful reference or two you can direct me to that may well correct my lack of understanding. Thirdly I think this a very likely useful case to preserve the raw diffraction images. All best wishes, John Prof John R Helliwell DSc On 19 Mar 2013, at 14:37, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model building (AutoBuild and ARP/wARP) but they cannot build anything that make some sense or build a random chains without any sense. I do not have an extensive knowledge of crystallography, but I have been thinking about some questions: If the third molecule (the bad one) is lying on the 2-fold symmetry axis on P 2 21 21, and since it does not have an intrinsic 2-fold symmetry axis (like protein molecule), how can I merge the structure factors (or intensities) related by symmetry and expand to lower symmetry afterwards? In this case the molecule lying on the 2-fold symmetry axis will have the structure factors wrongly merged, since the molecule is not symmetric, is it ok? If the third molecule is lying on the 2-fold symmetry axis on P 2 21 21, and only another two molecules can be related by the crystallographic symmetry, is it a case of pseudo-symmetry? But in this case, the third molecule is disordered in the crystal packing (as Zbyszek said), and probably have a long range disorder, because I cannot get a good maps for this third molecule even in P1. (pseudo-symmetry + order/disorder). And a more philosophical question… what is the problem in process data in a lower symmetry? Are there mathematical/statistical problems related that can lead to “false-good” data? I put a new .pdf file (ccp4bb_maps_P21.pdf) with map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps_P21.pdf I am sorry for so many questions and thanks in advance. Cheers, Andrey 2013/3/20 Jrh jrhelliw...@gmail.com Dear Zbyszek, I am concerned that the unmerged data would be bypassed and not preserved in your recommendation. I also find it counter intuitive that the merged data would then be unmerged into a lower symmetry and be better than the unmerged data; there is I imagine some useful reference or two you can direct me to that may well correct my lack of understanding. Thirdly I think this a very likely useful case to preserve the raw diffraction images. All best wishes, John Prof John R Helliwell DSc On 19 Mar 2013, at 14:37, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Edward, I think you gave a very good summary of what might have happened in the crystals. If the two domains diffract like a single crystal and interfere, F's are added, if they diffract like two different crystals, I's are added. In fact, one should model the special protein molecule in two alternative orientations, just like one does with individual amino acids that have multiple conformations. Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. Berry Sent: Tuesday, March 19, 2013 7:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Maybe this thread still needs some more pedagogy/explanation for those newbies and for biologist/ wanna-be crystallographers like me. My original reaction was- if the true space group is P21 you wouldn't want to expand from data reduced in higher symmetry, because you would be enforcing that higher symmetry. But if it were simply a case of P21 symmetry, with three molecules in the AU, that happened to have a beta angle of 90, merging statistics would have prevented reducing the data in p222 in the first place. Does order/disorder mean that the third molecule is actually present in two different orientations with equal occupancy, so that on the average it does obey the higher symmetry? Like our heme on a special position in the bacterioferritin paper? And structure factors add because the two orientations are present in the same domain, whereas with twinning the two orientations are present in different domains that diffract like separate crystals, and the resulting intensities add on the film? herman.schreu...@sanofi.com wrote: If it is crystal packing disorder (F's added instead of I's), the switches between the alternative conformations need to be very frequent, to be within coherent range, so I would asume that the alternative conformations will be present in equal proportions. Still the alternatives need to be modeled somehow and if this can be conveniently done in a lower symmetry spacegroup this would not artificially lower the free R-factors. As Phoebe mentioned, ignoring the higher symmetry relations and repicking the free Rflags at lower symmetry would lead free reflections to be linked to the working set, leading to too low Rfree values. However, with perfect packing disorder, no extra information would be gained by reprocessing in lower symmetry (in contrast to cases with pseudo symmetry). My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. Rice Sent: Tuesday, March 19, 2013 4:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree oops, I should have expanded my comments to include the sort of funky lattice order-disorder Zbyszek so cleverly diagnosed. Scratch that perfect twinning comment in my last message. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice [pr...@uchicago.edu] Sent: Tuesday, March 19, 2013 10:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Zbyszek, I am concerned that the unmerged data would be bypassed and not preserved in your recommendation. I also find it counter intuitive that the merged data would then be unmerged into a lower symmetry and be better than the unmerged data; there is I imagine some useful reference or two you can direct me to that may well correct my lack of understanding. Thirdly I think this a very likely useful case to preserve the raw diffraction images. All best wishes, John Prof John R Helliwell DSc On 19 Mar 2013, at 14:37, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey* Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey* Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Isn't lowering the symmetry equivalent to using multiple models/conformations for one map? I remember seeing this done with the infamous MSBA structure from a few years ago, so caveat emptor I guess. And further, wouldn't using strict NCS make things equivalent to the higher-symmetry space group? And then violating the NCS in places would then just be equivalent to modelling multiple conformations, no? JPK On Tue, Mar 19, 2013 at 11:34 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure
Re: [ccp4bb] Strange density in solvent channel and high Rfree
oops, I should have expanded my comments to include the sort of funky lattice order-disorder Zbyszek so cleverly diagnosed. Scratch that perfect twinning comment in my last message. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice [pr...@uchicago.edu] Sent: Tuesday, March 19, 2013 10:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive
Re: [ccp4bb] Strange density in solvent channel and high Rfree
If it is crystal packing disorder (F's added instead of I's), the switches between the alternative conformations need to be very frequent, to be within coherent range, so I would asume that the alternative conformations will be present in equal proportions. Still the alternatives need to be modeled somehow and if this can be conveniently done in a lower symmetry spacegroup this would not artificially lower the free R-factors. As Phoebe mentioned, ignoring the higher symmetry relations and repicking the free Rflags at lower symmetry would lead free reflections to be linked to the working set, leading to too low Rfree values. However, with perfect packing disorder, no extra information would be gained by reprocessing in lower symmetry (in contrast to cases with pseudo symmetry). My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. Rice Sent: Tuesday, March 19, 2013 4:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree oops, I should have expanded my comments to include the sort of funky lattice order-disorder Zbyszek so cleverly diagnosed. Scratch that perfect twinning comment in my last message. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice [pr...@uchicago.edu] Sent: Tuesday, March 19, 2013 10:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Maybe this thread still needs some more pedagogy/explanation for those newbies and for biologist/ wanna-be crystallographers like me. My original reaction was- if the true space group is P21 you wouldn't want to expand from data reduced in higher symmetry, because you would be enforcing that higher symmetry. But if it were simply a case of P21 symmetry, with three molecules in the AU, that happened to have a beta angle of 90, merging statistics would have prevented reducing the data in p222 in the first place. Does order/disorder mean that the third molecule is actually present in two different orientations with equal occupancy, so that on the average it does obey the higher symmetry? Like our heme on a special position in the bacterioferritin paper? And structure factors add because the two orientations are present in the same domain, whereas with twinning the two orientations are present in different domains that diffract like separate crystals, and the resulting intensities add on the film? herman.schreu...@sanofi.com wrote: If it is crystal packing disorder (F's added instead of I's), the switches between the alternative conformations need to be very frequent, to be within coherent range, so I would asume that the alternative conformations will be present in equal proportions. Still the alternatives need to be modeled somehow and if this can be conveniently done in a lower symmetry spacegroup this would not artificially lower the free R-factors. As Phoebe mentioned, ignoring the higher symmetry relations and repicking the free Rflags at lower symmetry would lead free reflections to be linked to the working set, leading to too low Rfree values. However, with perfect packing disorder, no extra information would be gained by reprocessing in lower symmetry (in contrast to cases with pseudo symmetry). My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. Rice Sent: Tuesday, March 19, 2013 4:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree oops, I should have expanded my comments to include the sort of funky lattice order-disorder Zbyszek so cleverly diagnosed. Scratch that perfect twinning comment in my last message. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice [pr...@uchicago.edu] Sent: Tuesday, March 19, 2013 10:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry
[ccp4bb] Strange density in solvent channel and high Rfree
*Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Check for translational NCS And you are way too conservative with resolution. Even those holding onto the Rmerge-dictated past would probably acquiesce to lower I/sig cutoff. If you are using aimless, follow its recommendations based on CC1/2, it's good for you. Cheers, Ed. On Fri, 2013-03-15 at 15:39 -0300, Andrey Nascimento wrote: Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Wow, Usually on the default settings of adxv, I can see spots at this I/sig! I suspect your data goes higher than 1.77. Include more of the high resolution data. You very likely don't have the correct space group. F On Mar 15, 2013, at 11:39 AM, Andrey Nascimento andreynascime...@gmail.com wrote: I/Isig.=4.4 - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Strange density in solvent channel and high Rfree
What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.com] Sent: Friday, March 15, 2013 1:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Hi Andrey, I am taking a risky guess: From your slide #2, it looks like a termini (unless this correspond to the beginning or end of disordered a loop) of the protein chain(s) is (are) extending toward(s) the solvent channel. Are the density features shown in slides #3,4, and 5 extend from this terminal residue?!! If this is true, did you missed to model any uncleaved-tag residues?!! I would also look at 2Fo-Fc at 1.0 sigma. Hope this helps, Partha On Fri, Mar 15, 2013 at 2:39 PM, Andrey Nascimento andreynascime...@gmail.com wrote: *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Or give it to arp/warp, either with the current model to improve or with phases from the current model to build new model? Phoebe A. Rice wrote: What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.com] *Sent:* Friday, March 15, 2013 1:39 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Strange density in solvent channel and high Rfree *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* ** *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
I second Phoebe's suggestion. Looks like another molecule to me. If you are doing molecular replacement you may need to get creative about trimming. When we had a case like that it wasn't until the third person worked on it that we go a solution because he trimmed the model differently than the first two who tried. Kendall On Mar 15, 2013, at 3:29 PM, Phoebe A. Rice pr...@uchicago.edumailto:pr...@uchicago.edu wrote: What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.commailto:andreynascime...@gmail.com] Sent: Friday, March 15, 2013 1:39 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey
