[ccp4bb] AW: [ccp4bb] Wrong Space Group?
Dear Bonsor, I fully second James suggestions but have a few additional comments: If you get a solution in P6522 with one molecule, you should get the same solution in P65 with 2 molecules. One of the crystallographic symmetry operators would then be non-crystallographic. The current version of Refmac will test all possible twinning operations, so there is no need to do it yourself (provided of course that you get a molecular replacement solution). I would also try your rebuilt model with extended helix as a model for MR. I suspect that the dimer which has formed is asymmetric and that it may be randomly packed in your crystal. If the helix is a small compared to the complete protein, it may not show up in twinning tests. Good luck! Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von James Holton Gesendet: Sonntag, 15. Dezember 2013 23:29 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Wrong Space Group? Its possible you are in a lower space group, perhaps with some twinning, but your search model is different enough to only find a solution when things are over-merged. Try refining your P6522 model against data merged in P65. If the other copy (symmetry mate in P6522) does not show up, you may be in trouble (wrong MR solution). I'd also try refinement/building in the other triogonal/hexagonal space groups, but again, start with the PDB file that you got for P6522. Just change the space group in the header, and switch out the MTZ file. You will need to merge your data in each space group and also check the a-b flip re-indexing for most of them. Have a look at the CCP4 reindexing list for the h,k,l operators to try: http://www.ccp4.ac.uk/html/reindexing.html note how similar they are to the twinning operators: http://www.ccp4.ac.uk/html/twinning.html If I have counted right, that means you have 36 jobs to run. I'd also recommend turning the TWIN option in refmac off and on for each of these cases. This will always give you a lower R factor, because of the dynamic range compression you get with twinning, but if one particular combination of twinning with a particular space group and axis reindexing is markedly better than all the others, then you have just found your right space group. So, now we are up to 72 jobs, but hardly a lot of work compared to growing the crystals in the first place. You might also want to try being clever and generating the symmetry mates of your P6522 model and refine these partners as separate molecules as you reduce the symmetry of the data. It's tricky, but think of it as an exercise. Which real-space operator becomes what reciprocal-space operator? You can check your answer by loading it up in coot and seeing if symmetry mates clash with the input coordinates. Yes, its a lot of work to try all these combinations, but that's the annoying thing about twinning, it opens up a lot of ambiguities. Good luck! -James Holton MAD Scientist On 12/14/2013 6:44 AM, D Bonsor wrote: Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can
Re: [ccp4bb] Wrong Space Group?
Its possible you are in a lower space group, perhaps with some twinning, but your search model is different enough to only find a solution when things are over-merged. Try refining your P6522 model against data merged in P65. If the other copy (symmetry mate in P6522) does not show up, you may be in trouble (wrong MR solution). I'd also try refinement/building in the other triogonal/hexagonal space groups, but again, start with the PDB file that you got for P6522. Just change the space group in the header, and switch out the MTZ file. You will need to merge your data in each space group and also check the a-b flip re-indexing for most of them. Have a look at the CCP4 reindexing list for the h,k,l operators to try: http://www.ccp4.ac.uk/html/reindexing.html note how similar they are to the twinning operators: http://www.ccp4.ac.uk/html/twinning.html If I have counted right, that means you have 36 jobs to run. I'd also recommend turning the TWIN option in refmac off and on for each of these cases. This will always give you a lower R factor, because of the dynamic range compression you get with twinning, but if one particular combination of twinning with a particular space group and axis reindexing is markedly better than all the others, then you have just found your right space group. So, now we are up to 72 jobs, but hardly a lot of work compared to growing the crystals in the first place. You might also want to try being clever and generating the symmetry mates of your P6522 model and refine these partners as separate molecules as you reduce the symmetry of the data. It's tricky, but think of it as an exercise. Which real-space operator becomes what reciprocal-space operator? You can check your answer by loading it up in coot and seeing if symmetry mates clash with the input coordinates. Yes, its a lot of work to try all these combinations, but that's the annoying thing about twinning, it opens up a lot of ambiguities. Good luck! -James Holton MAD Scientist On 12/14/2013 6:44 AM, D Bonsor wrote: Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can see that P6522 has no twin laws. Does that mean no twinning can occur in P6522 or that it can occur but there is no law to be able to separate the amplitudes? I also collected data on a single point mutation of the protein. Although this diffracted to a slightly weaker resolution (3.2Ang), I also observe the same problem of good maps in P6522 but no solution in the groups described above, a clear indication that this helix has elongated but terrible Rfactors. Based upon that the maps look good in P6522 do you believe that I have solved the structure in the correct space group but my data collection is at fault or in fact that I have some form of pseudosymmetry or something else going on and that the space group has lower symmetry but not in the space groups I have checked. Or is it something else. Any suggestions, criticisms or you need further information please contact me and enjoy your weekend.
Re: [ccp4bb] Wrong Space Group?
On Fri, 13 Dec 2013 19:44:44 +, D Bonsor dbon...@ihv.umaryland.edu wrote: Dear D, I agree with Tim and Jürgen that a) the map after Phaser, and before refinement is the most unbiased and should be used for sequence assignment. b) there may be a sequence register shift error that is responsible for the high R-values, and is masked by overfitting So I would try a) To stay on the safe side, you could chop the Phaser model into secondary structure elements and do rigid-body refinement. This yields maps that are largely unbiased. b) sharpening (very easy in coot) and inspection of these unbiased maps, to confirm the sequence register c) submit the Phaser model to Arp/wArp re-building, and also try the buccaneer/refmac iterative re-building, and maybe phenix.autobuild But the problem may also be your data. a) Maybe every second reflection is not integrated because it is weak? That is easy to check with XDSGUI using Tools/show frame with predicted spots b) other pathologies like spot overlap or experimental instability (what is the value of ISa in CORRECT.LP ?) - you could post FRAME.cbf and IDXREF.LP, INTEGRATE.LP and CORRECT.LP and pointless/xtriage statistics If the true space group is P6x22, then the data cannot be twinned. But if the true space group has lower symmetry the data may appear to be P6x22 . HTH, Kay P.S. XDSGUI latest version can be obtained from http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDSGUI Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can see that P6522 has no twin laws. Does that mean no twinning can occur in P6522 or that it can occur but there is no law to be able to separate the amplitudes? I also collected data on a single point mutation of the protein. Although this diffracted to a slightly weaker resolution (3.2Ang), I also observe the same problem of good maps in P6522 but no solution in the groups described above, a clear indication that this helix has elongated but terrible Rfactors. Based upon that the maps look good in P6522 do you believe that I have solved the structure in the correct space group but my data collection is at fault or in fact that I have some form of pseudosymmetry or something else going on and that the space group has lower symmetry but not in the space groups I have checked. Or is it something else. Any suggestions, criticisms or you need further information please contact me and enjoy your weekend.
[ccp4bb] Wrong Space Group?
Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can see that P6522 has no twin laws. Does that mean no twinning can occur in P6522 or that it can occur but there is no law to be able to separate the amplitudes? I also collected data on a single point mutation of the protein. Although this diffracted to a slightly weaker resolution (3.2Ang), I also observe the same problem of good maps in P6522 but no solution in the groups described above, a clear indication that this helix has elongated but terrible Rfactors. Based upon that the maps look good in P6522 do you believe that I have solved the structure in the correct space group but my data collection is at fault or in fact that I have some form of pseudosymmetry or something else going on and that the space group has lower symmetry but not in the space groups I have checked. Or is it something else. Any suggestions, criticisms or you need further information please contact me and enjoy your weekend.
Re: [ccp4bb] Wrong Space Group?
2.8 is not a terrible resolution to try out Buccaneer or Parrot. Your terrible R-factor might be due to a shift in residues perhaps ? Your After-building map does not show much of a side chain density to judge if you are in frame or off. But the elongated helix is in my eyes convincing enough. One thing you might want to try is to use composite omit maps to reduce your bias from MR and verify that what you built is indeed correct. Jürgen On Dec 13, 2013, at 2:44 PM, D Bonsor dbon...@ihv.umaryland.edumailto:dbon...@ihv.umaryland.edu wrote: Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can see that P6522 has no twin laws. Does that mean no twinning can occur in P6522 or that it can occur but there is no law to be able to separate the amplitudes? I also collected data on a single point mutation of the protein. Although this diffracted to a slightly weaker resolution (3.2Ang), I also observe the same problem of good maps in P6522 but no solution in the groups described above, a clear indication that this helix has elongated but terrible Rfactors. Based upon that the maps look good in P6522 do you believe that I have solved the structure in the correct space group but my data collection is at fault or in fact that I have some form of pseudosymmetry or something else going on and that the space group has lower symmetry but not in the space groups I have checked. Or is it something else. Any suggestions, criticisms or you need further information please contact me and enjoy your weekend. .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] wrong space group or wrong refinement strategy
If that solution applies to the original model you used (and not to re-solving the structure with the molecular replacement solution before or after refinement), then your tetramer model is just being rotated by 90 degrees around the 4-fold and placed on a different origin, i.e. the solution is equivalent to the starting model, except for applying symmetry and a change of origin. If that's true, then it implies that this is an isomorphous crystal to the one giving the model you're using for molecular replacement. Is that crystal in P41 with similar cell dimensions? Rigid-body refinement would be a sensible option in such a case, with the advantage that your new model is guaranteed to be on the same origin and thus easier to compare with the old model. Getting to the main question, you really have to look at the result of twinning tests. If they suggest that your crystal is twinned, then it's probably P41 with the twinning increasing the apparent symmetry of the data. But it's important to look at all the evidence, not just the Rfree after refinement, especially as the application of a twin target always lowers Rfree. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 1 Dec 2012, at 00:59, ruisher hu wrote: Hi, Dear CCP4 group, I recently collect one dataset and indexed as P4 space group. When I try to do MR with a tetramer as input, I found the solution file suggested P41. SOLU SET RFZ=5.2 TFZ=10.3 PAK=0 LLG=239 LLG=366 TFZ==20.9 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 50.265 0.217 219.800 FRAC 0.50029 0.49916 -0.00408 BFAC -0.04000 SOLU ENSE ensemble1 VRMS 0.639 SOLU SET RFZ=5.2 TFZ=10.0 PAK=0 LLG=239 TFZ==11.2 LLG=365 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 128.607 179.813 38.677 FRAC 0.49991 0.49905 -0.00208 BFAC -0.06264 SOLU ENSE ensemble1 VRMS 0.642 However when I did refinement in phenix, I have some trouble getting the R/Rfree down. It complains about some twinning or maybe higher symmetry like P422. When I apply twin law, h,-k,-l, I'm able to refine to 0.34/0.37 but still, hard to continue the refinement. The strategy I used for refinement is xyz coordinates, real-space, individual B-factors, occupancies, with NCS restraints and twin law. So I tried to rescaled into P41212 space group, and run MR again, with two chains as an input and found one solution SOLU SET RFZ=4.5 TFZ=9.5 PAK=0 LLG=115 TFZ==10.0 LLG=118 TFZ==10.4 SOLU SPAC P 41 21 2 SOLU 6DIM ENSE ET EULER 234.6 0.7 305.0 FRAC 1.00 0.49 -0.17 BFAC -0.14 Ensemble ET RMS variance(s): 0.96 However, when i tried to refine, the Rfree is high as 0.51 at the first round. Does this mean this is not the right solution or maybe some problems with the space group?Any suggestions for next step? Thanks very much.
[ccp4bb] wrong space group or wrong refinement strategy
Hi, Dear CCP4 group, I recently collect one dataset and indexed as P4 space group. When I try to do MR with a tetramer as input, I found the solution file suggested P41. SOLU SET RFZ=5.2 TFZ=10.3 PAK=0 LLG=239 LLG=366 TFZ==20.9 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 50.265 0.217 219.800 FRAC 0.50029 0.49916 -0.00408 BFAC -0.04000 SOLU ENSE ensemble1 VRMS 0.639 SOLU SET RFZ=5.2 TFZ=10.0 PAK=0 LLG=239 TFZ==11.2 LLG=365 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 128.607 179.813 38.677 FRAC 0.49991 0.49905 -0.00208 BFAC -0.06264 SOLU ENSE ensemble1 VRMS 0.642 However when I did refinement in phenix, I have some trouble getting the R/Rfree down. It complains about some twinning or maybe higher symmetry like P422. When I apply twin law, h,-k,-l, I'm able to refine to 0.34/0.37 but still, hard to continue the refinement. The strategy I used for refinement is xyz coordinates, real-space, individual B-factors, occupancies, with NCS restraints and twin law. So I tried to rescaled into P41212 space group, and run MR again, with two chains as an input and found one solution SOLU SET RFZ=4.5 TFZ=9.5 PAK=0 LLG=115 TFZ==10.0 LLG=118 TFZ==10.4 SOLU SPAC P 41 21 2 SOLU 6DIM ENSE ET EULER 234.6 0.7 305.0 FRAC 1.00 0.49 -0.17 BFAC -0.14 Ensemble ET RMS variance(s): 0.96 However, when i tried to refine, the Rfree is high as 0.51 at the first round. Does this mean this is not the right solution or maybe some problems with the space group?Any suggestions for next step? Thanks very much.
Re: [ccp4bb] wrong space group or wrong refinement strategy
Hello, What about the enantiomorphic space groups (the 4(3) screw axes instead of the 4(1) screw axes) ? These cannot be distinguised on the basis of structure factor amplitudes (unless you have an anomalous scatterer) nor on the basis of the specific extinctions (both screw axes extinguish the same reflections). However molecular replacement distinguishes between the two. I did not see any mention of these enantiomorphic space groups in your post hence my asking... Phaser allows to test everything. The CCP4 program Pointless is also good at letting you know what is the most likely space group. Hence you may have a combination of wrong space group and twinning. I can't really say with the information provided. I have had one case (also molecular replacement) where I had to test every single space group (a hexagonal lattice) in order to hit the correct one (that was before the days of Phaser). Also, with molecular replacement the first thing I do is to compare the initial R-f / R-free to the R-factors that are expected for a random distribution of atoms in the asymmetric unit (from memory, ca. 0.6). Once you have refined, the numbers go down so you can't really say anything any more. Furthermore, sometimes it is necessary to carry out the molecular replacement searches with a smaller set of atoms (for example when you are expecting to find a tetramer you carry out the searches with a dimer, or with a monomer - and sometimes you have the surprise to discover that the solvent content of your crystal is quite high and that the tetramer is formed by 2 dimers that are related by a crystallographic 2-fold axis - or the arrangement of molecules in your multimer is different from that in the search model, to sum it up: in crystallography, everything goes!). HTH, Fred. On 01/12/12 01:59, ruisher hu wrote: Hi, Dear CCP4 group, I recently collect one dataset and indexed as P4 space group. When I try to do MR with a tetramer as input, I found the solution file suggested P41. SOLU SET RFZ=5.2 TFZ=10.3 PAK=0 LLG=239 LLG=366 TFZ==20.9 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 50.265 0.217 219.800 FRAC 0.50029 0.49916 -0.00408 BFAC -0.04000 SOLU ENSE ensemble1 VRMS 0.639 SOLU SET RFZ=5.2 TFZ=10.0 PAK=0 LLG=239 TFZ==11.2 LLG=365 SOLU SPAC P 41 SOLU 6DIM ENSE ensemble1 EULER 128.607 179.813 38.677 FRAC 0.49991 0.49905 -0.00208 BFAC -0.06264 SOLU ENSE ensemble1 VRMS 0.642 However when I did refinement in phenix, I have some trouble getting the R/Rfree down. It complains about some twinning or maybe higher symmetry like P422. When I apply twin law, h,-k,-l, I'm able to refine to 0.34/0.37 but still, hard to continue the refinement. The strategy I used for refinement is xyz coordinates, real-space, individual B-factors, occupancies, with NCS restraints and twin law. So I tried to rescaled into P41212 space group, and run MR again, with two chains as an input and found one solution SOLU SET RFZ=4.5 TFZ=9.5 PAK=0 LLG=115 TFZ==10.0 LLG=118 TFZ==10.4 SOLU SPAC P 41 21 2 SOLU 6DIM ENSE ET EULER 234.6 0.7 305.0 tel:234.6%200.7%20305.0 FRAC 1.00 0.49 -0.17 BFAC -0.14 Ensemble ET RMS variance(s): 0.96 However, when i tried to refine, the Rfree is high as 0.51 at the first round. Does this mean this is not the right solution or maybe some problems with the space group?Any suggestions for next step? Thanks very much. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
