[ccp4bb] Twin refinement in Refmac
Dear colleagues, I am in the process of refining a P2(1) structure that is marked by Scala as twinned (L-test etc.) with a twinning fraction of ca. 0.2. I am still working on the twinning problem, so no more details about this. But what puzzles me right now is that, if I feed the data with a max. resolution of 1.95 A into Refmac with the twin refinement option on, it recognizes the resolution cut-off at first, but then uses a new cut-off of 1.88 A in the first ( and all subsequent) refinement cycles. Since there are no data at this resolution, this seems to screw up the calculation of R-values at high resolution and it reports for them zero-values in the co-ordinate header. Without twin refinement, the max. resolution stays at 1.95 A (but the final stats are worse, of course). Is this a bug or am I missing here something? Thanks in advance, and I am happy to send log files or my precious data if necessary. Cheers, Uli --- dr ulrich gohlke staff scientist - macromolecular structure and interaction max-delbrück-center for molecular medicine (mdc) +49 30 9406 - 2725 (w) +49 30 9406 - 2548 (fax) ulrich.goh...@mdc-berlin.demailto:ulrich.goh...@mdc-berlin.de http://www.mdc-berlin.de/en/research/research_teams/macromolecular_structure_and_interaction/
Re: [ccp4bb] Twin refinement in Refmac
Dear Uli, It seems that you are dealing with psued-twinning when cell do not overlap after rotation with twin operator. In these case what is in one resolution on one of the orientation becomes another resolution in another orientation of crystals In short:. You have an operator R that relates two orientation of the crystal. If R does not convert cell to itself completely then resolution of twin related reflections h' and h h' = Rh will be different. It usually means that in your images spots are split at higher resolution. If you have not integrated those spots as one at higher resolution then refinement may not be optimal. These cases needs to be considered as multiple orientation of the same crystal. I think MOSFLM should be able to integrate them as several cells. However refmac is not completely ready to deal with these cases as optimally as it should. I hope it clears things a bit. Cheers, Garib On 22 May 2014, at 15:48, ulrich.goh...@mdc-berlin.de ulrich.goh...@mdc-berlin.de wrote: Dear colleagues, I am in the process of refining a P2(1) structure that is marked by Scala as twinned (L-test etc.) with a twinning fraction of ca. 0.2. I am still working on the twinning problem, so no more details about this. But what puzzles me right now is that, if I feed the data with a max. resolution of 1.95 A into Refmac with the twin refinement option on, it recognizes the resolution cut-off at first, but then uses a new cut-off of 1.88 A in the first ( and all subsequent) refinement cycles. Since there are no data at this resolution, this seems to screw up the calculation of R-values at high resolution and it reports for them zero-values in the co-ordinate header. Without twin refinement, the max. resolution stays at 1.95 A (but the final stats are worse, of course). Is this a bug or am I missing here something? Thanks in advance, and I am happy to send log files or my precious data if necessary. Cheers, Uli --- dr ulrich gohlke staff scientist - macromolecular structure and interaction max-delbrück-center for molecular medicine (mdc) +49 30 9406 - 2725 (w) +49 30 9406 - 2548 (fax) ulrich.goh...@mdc-berlin.de http://www.mdc-berlin.de/en/research/research_teams/macromolecular_structure_and_interaction/ Dr Garib N Murshudov MRC-LMB Francis Crick Avenue Cambridge CB2 0QH UK Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Twin refinement in Refmac
Dear Garib, thanks for the clarification. I'll go back to the raw data and check for split spots at high resolution (and give Mosflm a try; initially, the data were integrated with XDS). Cheers, Uli --- dr ulrich gohlke staff scientist - macromolecular structure and interaction max-delbrück-center for molecular medicine (mdc) +49 30 9406 - 2725 (w) +49 30 9406 - 2548 (fax) ulrich.goh...@mdc-berlin.de http://www.mdc-berlin.de/en/research/research_teams/macromolecular_structure_and_interaction/
Re: [ccp4bb] twin refinement in refmac
Is this twinning or several crystals indexed according to different conventions? You usually see evidence of twinning for each crystal if it is really there.. Trigonal data can be indexed as h,k,l k,h,-k -h,-k,l or -k,-h,-l of course so you have a 75% chance of getting the 2nd crystal on a different convention than the first.. POINTLESS checks for this if you give it pairs of input files and does a good job in selecting the same indexing convention - providing of course the twinning isnt present. I would be suspicious because a) I have been convinced by having more than 2 twin domains b) when there is twinning the data analysis on each crystal seperately shows it up. Eleanor - On Mar 2 2012, wtempel wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] twin refinement in refmac
Hi all, I will like to know, as a follow up of what Prof. Randy Read said, what should be done to do the refinement against the measured data and not the detwinned F( which refmac outputs in the mtz after twin refinement), during subsequent refinements. And also, I would like to know how to ensure that the free R generated takes twinning into account if I am not using phenix. Thanks in advance, Regards, ARKO On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read rj...@cam.ac.uk wrote: I'm worried when you say that you use the initial job's output MTZ. Refmac replaces F with a detwinned F in the output file so you wouldn't be refining against your measured data in the subsequent round. Best wishes Randy Read Randy J. Read On 2 Mar 2012, at 02:00, wtempel wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
Re: [ccp4bb] twin refinement in refmac
Garib may have more to say, but the first point would be to always include the original data file as your input MTZ file for any cycle of refinement, whether you're using Refmac in CCP4 or phenix.refine. (In phenix.refine, if you assign the R-free data the first time you do refinement, it will produce a file containing all the information used in that cycle of refinement, including the new R-free flags and possibly any Hendrickson-Lattman coefficients, and you should use that file for all subsequent refinements.) As for whether R-free takes twinning into account, I think it's fair to say that all the refinement programs that handle twinning will do this appropriately. Regards, Randy Read On 2 Mar 2012, at 08:01, arka chakraborty wrote: Hi all, I will like to know, as a follow up of what Prof. Randy Read said, what should be done to do the refinement against the measured data and not the detwinned F( which refmac outputs in the mtz after twin refinement), during subsequent refinements. And also, I would like to know how to ensure that the free R generated takes twinning into account if I am not using phenix. Thanks in advance, Regards, ARKO On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read rj...@cam.ac.uk wrote: I'm worried when you say that you use the initial job's output MTZ. Refmac replaces F with a detwinned F in the output file so you wouldn't be refining against your measured data in the subsequent round. Best wishes Randy Read Randy J. Read On 2 Mar 2012, at 02:00, wtempel wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel -- ARKA CHAKRABORTY CAS in Crystallography and Biophysics University of Madras Chennai,India -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 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
Re: [ccp4bb] twin refinement in refmac
1) You should use measured data (after scala/aimless/truncate). In general there may not be one to one relationship between observed data and asymmetric unit (e.g. non-merohedral twinning) and it would not be possible to bring input data to output file. Use original data 2) Internally refmac groups reflection into classes. All twin related reflections belong to one class. In the example you give four reflections belong to one class. FreeR is property of the class not individual relfections. I.e. all twin related reflections belong either to free or working class. If your input data has this property then output should also have this. In the output file there will be 0 (for free) and 1 (working) 3) At early stages it is not easy to factorise twin and underestimated symmetry. It seems that L-test is the best way of making decision if you crystals are twinnined. If you collect data from many crystals and merge them then you artificially twin (or increase twinning). Using pointless to index all datasets consistently may sort some of the problems 4) In this case it seems that you may need to reindex. Largest twin domain is not the first one Regards Garib On 2 Mar 2012, at 02:00, wtempel wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] twin refinement in refmac
On 2 Mar 2012, at 08:01, arka chakraborty wrote: Hi all, I will like to know, as a follow up of what Prof. Randy Read said, what should be done to do the refinement against the measured data and not the detwinned F( which refmac outputs in the mtz after twin refinement), during subsequent refinements. Operationally, don't put in the MTZ in field of the GUI something that Refmac generated for you in a previous run as MTZ out file. Always use as MTZ in your original data file. And also, I would like to know how to ensure that the free R generated takes twinning into account if I am not using phenix. Refmac does take in consideration the twin law(s) when handling free reflections . This is the case even if you have generated your free reflections randomly. Internally Refmac will modify your Free set in such a way that twin related reflections are in the same group (free or working) -- classes mentioned by Garib The good thing about this is that twin-related reflections are handled properly during refinement irrespective of your Free-set choice. The bad thing (Garib please correct me if I am wrong here) is that you might end up depositing a Free set which is not that actually used in refinement. Best wishes Roberto Thanks in advance, Regards, ARKO On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read rj...@cam.ac.ukmailto:rj...@cam.ac.uk wrote: I'm worried when you say that you use the initial job's output MTZ. Refmac replaces F with a detwinned F in the output file so you wouldn't be refining against your measured data in the subsequent round. Best wishes Randy Read Randy J. Read On 2 Mar 2012, at 02:00, wtempel wtem...@gmail.commailto:wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel -- ARKA CHAKRABORTY CAS in Crystallography and Biophysics University of Madras Chennai,India Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.ukmailto:roberto.stei...@kcl.ac.uk
Re: [ccp4bb] twin refinement in refmac
Hi all, Thanks for the express replies. Your insights along with the article by Prof. Garib pointed to by Prof. Pavel completes the story for me. Regards, ARKO On Fri, Mar 2, 2012 at 3:09 PM, Steiner, Roberto roberto.stei...@kcl.ac.ukwrote: On 2 Mar 2012, at 08:01, arka chakraborty wrote: Hi all, I will like to know, as a follow up of what Prof. Randy Read said, what should be done to do the refinement against the measured data and not the detwinned F( which refmac outputs in the mtz after twin refinement), during subsequent refinements. Operationally, don't put in the MTZ in field of the GUI something that Refmac generated for you in a previous run as MTZ out file. Always use as MTZ in your original data file. And also, I would like to know how to ensure that the free R generated takes twinning into account if I am not using phenix. Refmac does take in consideration the twin law(s) when handling free reflections . This is the case even if you have generated your free reflections randomly. Internally Refmac will modify your Free set in such a way that twin related reflections are in the same group (free or working) -- classes mentioned by Garib The good thing about this is that twin-related reflections are handled properly during refinement irrespective of your Free-set choice. The bad thing (Garib please correct me if I am wrong here) is that you might end up depositing a Free set which is not that actually used in refinement. Best wishes Roberto Thanks in advance, Regards, ARKO On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read rj...@cam.ac.uk wrote: I'm worried when you say that you use the initial job's output MTZ. Refmac replaces F with a detwinned F in the output file so you wouldn't be refining against your measured data in the subsequent round. Best wishes Randy Read Randy J. Read On 2 Mar 2012, at 02:00, wtempel wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India* Roberto Steiner, PhD Group Leader Randall Division of Cell and Molecular Biophysics King's College London Room 3.10A New Hunt's House Guy's Campus SE1 1UL, London, UK Tel 0044-20-78488216 Fax 0044-20-78486435 roberto.stei...@kcl.ac.uk -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
[ccp4bb] twin refinement in refmac
Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel
Re: [ccp4bb] twin refinement in refmac
Hi Wolfram, a few points: - R-factors in twin refinement vs non-twin refinement are not directly comparable: G.N. Murshudov, Appl. Comput. Math., V.10, N.2, 2011, pp.250-261 http://www.science.az/acm/V10,%20N2,%202011,%20pdf/250-261.pdf - did you make sure free-R flags assigned having twinning in mind (what phenix.refine always does)? Otherwise you have a risk of having this: see page 14 here: http://phenix-online.org/presentations/latest/pavel_validation.pdf Pavel On Thu, Mar 1, 2012 at 6:00 PM, wtempel wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel
Re: [ccp4bb] twin refinement in refmac
I'm worried when you say that you use the initial job's output MTZ. Refmac replaces F with a detwinned F in the output file so you wouldn't be refining against your measured data in the subsequent round. Best wishes Randy Read Randy J. Read On 2 Mar 2012, at 02:00, wtempel wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel
