[ccp4bb] Twinning problem
Dear Bulletin Board, Prodded by pdb annotators, which are very hesitant to accept coordinate files when their Rfactor does not correspond with our Rfactor, I had a look again into some old data sets, which I suspect are twinned. Below are the results of some twinning tests with the Detwin program (top value: all reflections, lower value: reflections > Nsig*obs (whatever that may mean). The space group is P32, the resolution is 2.3 - 2.6 Å and data are reasonable complete: 95 - 100%. >From the Detwin analysis, it seems that the crystals are twinned with twin >operator k,h,-l with a twinning fraction of 0.3 for crystal 1, 0.15 for >crystal 2 and 0.4 for crystal 3. Crystal 2 can be refined while ignoring >twinning to get acceptable but not stellar R and Rfree values. However, when I >try to detwin Fobs of e.g. crystal 1 (twinning fraction 0.3), R and Rfree >values stay about the same, whatever twinning fraction I try. At the time, I >used the CNS detwin_perfect protocol to detwin using Fcalcs, which brought the >Rfactors in acceptable range, but I do not feel that was the perfect solution. >Ignoring twinning on e.g. crystal 1 produces an Rfactor of 22% and an Rfree of >29% Do you have any idea what could be going on? Thank you for your help! Herman Crystal 1: operator -h,-k,l Suggests Twinning factor (0.5-H):0.113 Suggests Twinning factor (0.5-H):0.147 operator: k,h,-l Suggests Twinning factor (0.5-H):0.277 Suggests Twinning factor (0.5-H):0.323 operator -k,-h,-l Suggests Twinning factor (0.5-H):0.101 Suggests Twinning factor (0.5-H):0.134 Crystal 2: operator -h,-k,l Suggests Twinning factor (0.5-H):0.077 Suggests Twinning factor (0.5-H):0.108 operator: k,h,-l Suggests Twinning factor (0.5-H):0.126 Suggests Twinning factor (0.5-H):0.161 operator -k,-h,-l Suggests Twinning factor (0.5-H):0.072 Suggests Twinning factor (0.5-H):0.106 Crystal 3: operator -h,-k,l Suggests Twinning factor (0.5-H):0.123 Suggests Twinning factor (0.5-H):0.149 operator: k,h,-l Suggests Twinning factor (0.5-H):0.393 Suggests Twinning factor (0.5-H):0.433 operator -k,-h,-l Suggests Twinning factor (0.5-H):0.110 Suggests Twinning factor (0.5-H):0.133
[ccp4bb] twinning problem ?
Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions Stephen -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email: cus...@embl.fr Website: http://www.embl.fr Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 6 Rue Jules Horowitz, 38042 Grenoble, France **
[ccp4bb] Twinning problem
To the CCP4 community, I have collected data from an RNA molecule that extends to 2.9 angstroms, exhibit mosaicity less than 0.9 degrees and generally show nice, round spots. The crystals look cubic and are not birefringent (suggesting a cubic lattice). However, the data index poorly with the best solutions being either I23 (a=b=c=141.68) or I422 (a=b=141.74; c=141.57). Predicting reflections using each of these indexing solutions appears to confirm each as a valid indexing solution. However regardless of which space group I select to process the data, the final result after scaling reveals relatively high Rsym values overall and for individual batches (10% - 20%). Also, a large wedge of data are required to achieve nearly 100% completeness (>60 degrees; if the lattice was truly cubic I would expect much less to be required for high completeness). These discrepancies led a colleague to suggest twinning might be a problem. The UCLA twinning server (Yeates method) finds the following: For the data processed as I422 in the partial twinning test: No merohedral twinning laws found for that space group For the data processed as I23 in the partial twinning test: Twin fraction of 0.408 For the data processed as I23 in the perfect merohedral twinning test: Resolution ; / 2 16.147 ; 1.99 (n = 404) 7.224 ; 1.57 (n = 404) 6.067 ; 1.40 (n = 404) 5.434 ; 1.33 (n = 404) 5.006 ; 1.37 (n = 404) 4.687 ; 1.44 (n = 404) 4.440 ; 1.35 (n = 404) 4.240 ; 1.34 (n = 404) 4.070 ; 1.88 (n = 404) 3.924 ; 1.54 (n = 404) 3.799 ; 1.61 (n = 404) 3.689 ; 2.03 (n = 404) 3.590 ; 1.74 (n = 404) 3.501 ; 2.00 (n = 404) 3.421 ; 2.08 (n = 404) 3.347 ; 2.17 (n = 404) 3.278 ; 2.15 (n = 404) 3.216 ; 1.42 (n = 404) 3.158 ; 1.58 (n = 404) 3.104 ; 1.69 (n = 404) 3.054 ; 1.59 (n = 404) 3.007 ; 1.48 (n = 404) 2.962 ; 1.80 (n = 404) 2.920 ; 5.42 (n = 404) This is my first experience with twinning (hmmm...I feel like I'm being initiated), and I have several questions that I have not been able to answer yet from researching the literature or CCP4bb archives. I should mention that several data sets from several similar crystals all behave the same in terms of the difficulties in data reduction and even the apparent twinning fraction (the same to within a few %). I know the first advice will be to try new conditions, but I wonder if I can work with these data since I already collected data sets for several derivatives and also anomalous. Any advice or literature references are greatly appreciated to any or all of these questions: 1.) How should I go about assigning/identifying the correct space group? Does the apparent presence of merohedral twinning for the I23 processed data, but not I422, indicated that I do not have a cubic lattice? 2.) How is it possible that the data processed in the lower symmetry I422 space group are not also found to be twinned? I can't visualize how the same merohedrally twinned lattice could be described without conflict in the lower symmetry space group. 3.) I looked at the original T. Yeates paper in Meth. Enz. regarding twinning. There is an example of data from plastocyanin which are perfectly twinned. The reported plot of / squared as a function of resolution show a fluctuation around 1.5 that looks similar to the values I reported above as output from the perfect twinning test. How does one determine from those plots whether or not you have perfect merohedral twinning? Should I consider the average value, the lowest value, the distribution, or is my apparent partial twinning fraction sufficiently far from 50% to be sure that I don't? 4.) I tried running the perfect- and partial-merohedral detwinning scripts in CNS for the data processed as the I23 space group. The result of the perfect-merohedral detwinning script resulted in generally higher values of / squared, but it's not clear to me what that means or how it is possible to detwin perfect merohedral twinned data. After the partial-merohedral detwinning script however, the twinning fraction dropped to 17%. Is that informative with regards to what space group I'm dealing with or whether or not I have partial vs. perfect twinning? 5.) The last questions are about how to proceed with solving the structure. As I mentioned, I have collected data that I hope to use for MIR, potentially including anomalous. With a twinning fraction of 17% after detwinning, is it possible/appropriate to solve the structure by MIR or SIRAS (I'm guessing differences in the twinning will just diminish my signal to noise for finding the heavy atom peaks)? I also understand that it is possible to solve a perfectly merohedrally twinned data set by molecular replacement. I have a partial MR solution using the I23 data that appears to have unique phase information. I know there are several refinement programs that could be used for twinned data. Can anyone recommend one that handles RNA well? Thank you very
[ccp4bb] Twinning problem
Hi, All, I got a set of P2(or P21) data for MR. However, the Phenix-Xtriage indicated that it could be a pseudo-merohedral twinning. Does anyone know how to deal with such kind of twinning problem? Thanks. Best, Liang
Re: [ccp4bb] Twinning problem
You are welcome. Let me also for the benefit of others who may search the archives in the future, let me correct two errors below - (typo and a miss-recollection). Specially, I was thinking that phenix.refine was now able to refine multiple twin laws, but according to Nat Echols on the phenix mailing list http://phenix-online.org/pipermail/phenixbb/2013-March/019538.html phenix.refine only handles 1 twin law at this time. (My typo was that and our second structure was 3nuz with twin fractions 0.38, 0.32, 0.16 and 0.14 -- not 2nuz). A useful search for deposited structures mentioning tetartohedral http://www.ebi.ac.uk/pdbe-srv/view/search?search_type=all_text&text=TETARTOHEDRALLY+OR+TETARTOHEDRAL Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.com Sent: Thursday, June 20, 2013 8:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] AW: Twinning problem Dear Mitch (and Philip and Phil), It is clear that I should give refmac a go with the non-detwinned F's and just the TWIN command. Thank you for your suggestions, Herman -Ursprüngliche Nachricht- Von: Miller, Mitchell D. [mailto:mmil...@slac.stanford.edu] Gesendet: Donnerstag, 20. Juni 2013 16:18 An: Schreuder, Herman R&D/DE Betreff: RE: Twinning problem Hi Herman, Have you considered the possibility of your crystals being tetartohedral twinned. That is more than one of the twin laws may apply to your crystals. E.g. in P32 it is possible to have tetartohedral twinning which would have 4 twin domains - (h,k,l), (k,h,-l), (-h,-k,l) and (-k,-h,-l). Perfect tetartohedral twinning of P3 would merge in P622 and each twin domain would have a faction of 0.25. We have had 2 cases like this (the first 2PRX was before there was support for this type of twinning except for in shelxl and we ended up with refined twin fractions of 0.38, 0.28, 0.19, 0.15 for the deposited crystal and a 2nd crystal that we did not deposit had twin fractions of 0.25, 0.27, 0.17, 0.31). The 2nd case we had was after support for twining (including tetartohedral twinning) was added to refmac (and I think phenix.refine can also handle this). For 2NUZ, it was P32 with refined twin fractions of 0.25, 0.27, 0.17, 0.31. Pietro Roversi wrote a review of tetartohedral twinning for the CCP4 proceedings issues of acta D http://dx.doi.org/10.1107/S0907444912006737 I would try refinement with refmac using the original (non-detwinned F's) with just the TWIN command to see if it ends up keeping twin fractions for all 3 operators (4 domains) -- especially with crystals 1 and 3 which appear to have the largest estimates of the other twin fractions. Regards, Mitch == Mitchell Miller, Ph.D. Joint Center for Structural Genomics Stanford Synchrotron Radiation Lightsource 2575 Sand Hill Rd -- SLAC MS 99 Menlo Park, CA 94025 Phone: 1-650-926-5036 FAX: 1-650-926-3292 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.com Sent: Thursday, June 20, 2013 6:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Twinning problem Dear Bulletin Board, Prodded by pdb annotators, which are very hesitant to accept coordinate files when their Rfactor does not correspond with our Rfactor, I had a look again into some old data sets, which I suspect are twinned. Below are the results of some twinning tests with the Detwin program (top value: all reflections, lower value: reflections > Nsig*obs (whatever that may mean). The space group is P32, the resolution is 2.3 - 2.6 Å and data are reasonable complete: 95 - 100%. >From the Detwin analysis, it seems that the crystals are twinned with twin >operator k,h,-l with a twinning fraction of 0.3 for crystal 1, 0.15 for >crystal 2 and 0.4 for crystal 3. Crystal 2 can be refined while ignoring >twinning to get acceptable but not stellar R and Rfree values. However, when I >try to detwin Fobs of e.g. crystal 1 (twinning fraction 0.3), R and Rfree >values stay about the same, whatever twinning fraction I try. At the time, I >used the CNS detwin_perfect protocol to detwin using Fcalcs, which brought the >Rfactors in acceptable range, but I do not feel that was the perfect solution. >Ignoring twinning on e.g. crystal 1 produces an Rfactor of 22% and an Rfree of >29% Do you have any idea what could be going on? Thank you for your help! Herman Crystal 1: operator -h,-k,l Suggests Twinning factor (0.5-H):0.113 Suggests Twinning factor (0.5-H):0.147 operator: k,h,-l Suggests Twinning factor (0.5-H):0.277 Suggests Twinning factor (0.5-H):0.323 operator -k,-h,-l Suggests Twinning factor (0.5-H):0.101 Suggests Twinning factor (0.5-H):0.134 Crystal 2: operator -h,-k,l Suggests Twinning factor (0.5-H):0.077 Suggests T
Re: [ccp4bb] twinning problem ?
What is the NC translation? If there is a factor of 0.5 that makes SG determination complicated.. Eleanor On 11 March 2014 14:04, Stephen Cusack wrote: > Dear All, > I have 2.6 A data and unambiguous molecular replacement solution for > two copies/asymmetric unit of a 80 K protein for a crystal integrated > in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. > Refinement allowed rebuilding/completion of the model in the noraml way > but the R-free does not go below 30%. The map in the model regions looks > generally fine but there is a lot > of extra positive density in the solvent regions (some of it looking like > weak density for helices and strands) and unexpected positive peaks within > the model region. > Careful inspection allowed manual positioning of a completely different, > overlapping solution for the dimer which fits the extra density perfectly. > The two incompatible solutions are related by a 2-fold axis parallel to a. > This clearly suggests some kind of twinning. However twinning analysis > programmes (e.g. Phenix-Xtriage), while suggesting the potentiality > of pseudo-merohedral twinning (-h, l, k) do not reveal > any significant twinning fraction and proclaim the data likely to be > untwinned. (NB. The programmes do however highlight a > non-crystallographic translation and there are systematic intensity > differences in the data). Refinement, including this twinning law made no > difference > since the estimated twinning fraction was 0.02. Yet the extra density is > clearly there and I know exactly the real-space transformation between the > two packing solutions. > How can I best take into account this alternative solution (occupancy > seems to be around 20-30%) in the refinement ? > thanks for your suggestions > Stephen > > -- > > ** > Dr. Stephen Cusack, > Head of Grenoble Outstation of EMBL > Group leader in structural biology of protein-RNA complexes and viral > proteins > Joint appointment in EMBL Genome Biology Programme > Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host > Cell Interactions (UVHCI) > ** > > Email: cus...@embl.fr > Website: http://www.embl.fr > Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 > > Fax:(33) 4 76 20 7199 > Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, > 38042 Grenoble Cedex 9, France > Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, > 6 Rue Jules Horowitz, 38042 Grenoble, France > ** >
Re: [ccp4bb] twinning problem ?
Sorry - hadnt finished.. The twinning tests are distorted by NC translation - usually the L test is safe, but the others are all suspect.. On 11 March 2014 14:09, Eleanor Dodson wrote: > What is the NC translation? If there is a factor of 0.5 that makes SG > determination complicated.. > Eleanor > > > On 11 March 2014 14:04, Stephen Cusack wrote: > >> Dear All, >> I have 2.6 A data and unambiguous molecular replacement solution for >> two copies/asymmetric unit of a 80 K protein for a crystal integrated >> in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. >> Refinement allowed rebuilding/completion of the model in the noraml way >> but the R-free does not go below 30%. The map in the model regions looks >> generally fine but there is a lot >> of extra positive density in the solvent regions (some of it looking like >> weak density for helices and strands) and unexpected positive peaks within >> the model region. >> Careful inspection allowed manual positioning of a completely different, >> overlapping solution for the dimer which fits the extra density perfectly. >> The two incompatible solutions are related by a 2-fold axis parallel to a. >> This clearly suggests some kind of twinning. However twinning analysis >> programmes (e.g. Phenix-Xtriage), while suggesting the potentiality >> of pseudo-merohedral twinning (-h, l, k) do not reveal >> any significant twinning fraction and proclaim the data likely to be >> untwinned. (NB. The programmes do however highlight a >> non-crystallographic translation and there are systematic intensity >> differences in the data). Refinement, including this twinning law made no >> difference >> since the estimated twinning fraction was 0.02. Yet the extra density is >> clearly there and I know exactly the real-space transformation between the >> two packing solutions. >> How can I best take into account this alternative solution (occupancy >> seems to be around 20-30%) in the refinement ? >> thanks for your suggestions >> Stephen >> >> -- >> >> ** >> Dr. Stephen Cusack, >> Head of Grenoble Outstation of EMBL >> Group leader in structural biology of protein-RNA complexes and viral >> proteins >> Joint appointment in EMBL Genome Biology Programme >> Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host >> Cell Interactions (UVHCI) >> ** >> >> Email: cus...@embl.fr >> Website: http://www.embl.fr >> Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 >> >> Fax:(33) 4 76 20 7199 >> Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, >> 38042 Grenoble Cedex 9, France >> Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, >> 6 Rue Jules Horowitz, 38042 Grenoble, France >> ** >> > >
Re: [ccp4bb] twinning problem ?
Hi, If there's an NCS translation, recent versions of Phaser can account for it and give moment tests that can detect twinning even in the presence of tNCS. But I agree with Eleanor that the L test is generally a good choice in these cases. However, the fact that you see density suggests that your crystal might be more on the statistical disorder side of the statistical disorder <--> twinning continuum, i.e. the crystal doesn't have mosaic blocks corresponding to one twin fraction that are large compared to the coherence length of the X-rays. So you might want to try refinement with the whole structure duplicated as alternate conformers. 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 11 Mar 2014, at 14:10, Eleanor Dodson wrote: > Sorry - hadnt finished.. > The twinning tests are distorted by NC translation - usually the L test is > safe, but the others are all suspect.. > > > > On 11 March 2014 14:09, Eleanor Dodson wrote: > What is the NC translation? If there is a factor of 0.5 that makes SG > determination complicated.. > Eleanor > > > On 11 March 2014 14:04, Stephen Cusack wrote: > Dear All, > I have 2.6 A data and unambiguous molecular replacement solution for two > copies/asymmetric unit of a 80 K protein for a crystal integrated > in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. > Refinement allowed rebuilding/completion of the model in the noraml way but > the R-free does not go below 30%. The map in the model regions looks > generally fine but there is a lot > of extra positive density in the solvent regions (some of it looking like > weak density for helices and strands) and unexpected positive peaks within > the model region. > Careful inspection allowed manual positioning of a completely different, > overlapping solution for the dimer which fits the extra density perfectly. > The two incompatible solutions are related by a 2-fold axis parallel to a. > This clearly suggests some kind of twinning. However twinning analysis > programmes (e.g. Phenix-Xtriage), while suggesting the potentiality > of pseudo-merohedral twinning (-h, l, k) do not reveal > any significant twinning fraction and proclaim the data likely to be > untwinned. (NB. The programmes do however highlight a > non-crystallographic translation and there are systematic intensity > differences in the data). Refinement, including this twinning law made no > difference > since the estimated twinning fraction was 0.02. Yet the extra density is > clearly there and I know exactly the real-space transformation between the > two packing solutions. > How can I best take into account this alternative solution (occupancy seems > to be around 20-30%) in the refinement ? > thanks for your suggestions > Stephen > > -- > > ** > Dr. Stephen Cusack, > Head of Grenoble Outstation of EMBL > Group leader in structural biology of protein-RNA complexes and viral proteins > Joint appointment in EMBL Genome Biology Programme > Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell > Interactions (UVHCI) > ** > > Email: cus...@embl.fr > Website: http://www.embl.fr > Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 > > Fax:(33) 4 76 20 7199 > Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, > 38042 Grenoble Cedex 9, France > Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, > 6 Rue Jules Horowitz, 38042 Grenoble, France > ** > >
Re: [ccp4bb] twinning problem ?
Shape of the diffraction spots changes in the statistical disorder <--> > twinning continuum. At both ends spots shape is like in diffraction from crystals without such disorder. However, in the intermediate case, electron density autocorrelation function has additional component to one resulting from ordered crystal. This additional component of autocorrelation creates characteristic non-Bragg diffraction, e.g. streaks aligned with particular unit cell axis. In the absence of such diffraction pattern, the ambiguity is binary. The description of the problem indicates statistical disorder. Zbyszek Otwinowski > Hi, > > If there's an NCS translation, recent versions of Phaser can account for > it and give moment tests that can detect twinning even in the presence of > tNCS. But I agree with Eleanor that the L test is generally a good choice > in these cases. > > However, the fact that you see density suggests that your crystal might be > more on the statistical disorder side of the statistical disorder <--> > twinning continuum, i.e. the crystal doesn't have mosaic blocks > corresponding to one twin fraction that are large compared to the > coherence length of the X-rays. So you might want to try refinement with > the whole structure duplicated as alternate conformers. > > 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 Road > E-mail: rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > On 11 Mar 2014, at 14:10, Eleanor Dodson > wrote: > >> Sorry - hadnt finished.. >> The twinning tests are distorted by NC translation - usually the L test >> is safe, but the others are all suspect.. >> >> >> >> On 11 March 2014 14:09, Eleanor Dodson >> wrote: >> What is the NC translation? If there is a factor of 0.5 that makes SG >> determination complicated.. >> Eleanor >> >> >> On 11 March 2014 14:04, Stephen Cusack wrote: >> Dear All, >> I have 2.6 A data and unambiguous molecular replacement solution for >> two copies/asymmetric unit of a 80 K protein for a crystal >> integrated >> in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. >> Refinement allowed rebuilding/completion of the model in the noraml way >> but the R-free does not go below 30%. The map in the model regions looks >> generally fine but there is a lot >> of extra positive density in the solvent regions (some of it looking >> like weak density for helices and strands) and unexpected positive >> peaks within the model region. >> Careful inspection allowed manual positioning of a completely different, >> overlapping solution for the dimer which fits the extra density >> perfectly. >> The two incompatible solutions are related by a 2-fold axis parallel to >> a. >> This clearly suggests some kind of twinning. However twinning analysis >> programmes (e.g. Phenix-Xtriage), while suggesting the potentiality >> of pseudo-merohedral twinning (-h, l, k) do not reveal >> any significant twinning fraction and proclaim the data likely to be >> untwinned. (NB. The programmes do however highlight a >> non-crystallographic translation and there are systematic intensity >> differences in the data). Refinement, including this twinning law made >> no difference >> since the estimated twinning fraction was 0.02. Yet the extra density is >> clearly there and I know exactly the real-space transformation between >> the two packing solutions. >> How can I best take into account this alternative solution (occupancy >> seems to be around 20-30%) in the refinement ? >> thanks for your suggestions >> Stephen >> >> -- >> >> ** >> Dr. Stephen Cusack, >> Head of Grenoble Outstation of EMBL >> Group leader in structural biology of protein-RNA complexes and viral >> proteins >> Joint appointment in EMBL Genome Biology Programme >> Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host >> Cell Interactions (UVHCI) >> ** >> >> Email: cus...@embl.fr >> Website: http://www.embl.fr >> Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 >> Fax:(33) 4 76 20 7199 >> Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, >> 38042 Grenoble Cedex 9, France >> Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, >> 6 Rue Jules Horowitz, 38042 Grenoble, France >> ** >> >> > 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] twinning problem ?
Zbyszek - do you have any measure of unintegrated streaks? It could be a help to at least have a rough score. Eleanor On 11 March 2014 20:04, Zbyszek Otwinowski wrote: > Shape of the diffraction spots changes in the statistical disorder <--> > > twinning continuum. At both ends spots shape is like in diffraction from > crystals without such disorder. However, in the intermediate case, > electron density autocorrelation function has additional component to > one resulting from ordered crystal. This additional component of > autocorrelation creates characteristic non-Bragg diffraction, e.g. > streaks aligned with particular unit cell axis. > > In the absence of such diffraction pattern, the ambiguity is binary. The > description of the problem indicates statistical disorder. > > Zbyszek Otwinowski > > > Hi, > > > > If there's an NCS translation, recent versions of Phaser can account for > > it and give moment tests that can detect twinning even in the presence of > > tNCS. But I agree with Eleanor that the L test is generally a good > choice > > in these cases. > > > > However, the fact that you see density suggests that your crystal might > be > > more on the statistical disorder side of the statistical disorder <--> > > twinning continuum, i.e. the crystal doesn't have mosaic blocks > > corresponding to one twin fraction that are large compared to the > > coherence length of the X-rays. So you might want to try refinement with > > the whole structure duplicated as alternate conformers. > > > > 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 Road > > E-mail: rj...@cam.ac.uk > > Cambridge CB2 0XY, U.K. > > www-structmed.cimr.cam.ac.uk > > > > On 11 Mar 2014, at 14:10, Eleanor Dodson > > wrote: > > > >> Sorry - hadnt finished.. > >> The twinning tests are distorted by NC translation - usually the L test > >> is safe, but the others are all suspect.. > >> > >> > >> > >> On 11 March 2014 14:09, Eleanor Dodson > >> wrote: > >> What is the NC translation? If there is a factor of 0.5 that makes SG > >> determination complicated.. > >> Eleanor > >> > >> > >> On 11 March 2014 14:04, Stephen Cusack wrote: > >> Dear All, > >> I have 2.6 A data and unambiguous molecular replacement solution for > >> two copies/asymmetric unit of a 80 K protein for a crystal > >> integrated > >> in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. > >> Refinement allowed rebuilding/completion of the model in the noraml way > >> but the R-free does not go below 30%. The map in the model regions looks > >> generally fine but there is a lot > >> of extra positive density in the solvent regions (some of it looking > >> like weak density for helices and strands) and unexpected positive > >> peaks within the model region. > >> Careful inspection allowed manual positioning of a completely different, > >> overlapping solution for the dimer which fits the extra density > >> perfectly. > >> The two incompatible solutions are related by a 2-fold axis parallel to > >> a. > >> This clearly suggests some kind of twinning. However twinning analysis > >> programmes (e.g. Phenix-Xtriage), while suggesting the potentiality > >> of pseudo-merohedral twinning (-h, l, k) do not reveal > >> any significant twinning fraction and proclaim the data likely to be > >> untwinned. (NB. The programmes do however highlight a > >> non-crystallographic translation and there are systematic intensity > >> differences in the data). Refinement, including this twinning law made > >> no difference > >> since the estimated twinning fraction was 0.02. Yet the extra density is > >> clearly there and I know exactly the real-space transformation between > >> the two packing solutions. > >> How can I best take into account this alternative solution (occupancy > >> seems to be around 20-30%) in the refinement ? > >> thanks for your suggestions > >> Stephen > >> > >> -- > >> > >> ** > >> Dr. Stephen Cusack, > >> Head of Grenoble Outstation of EMBL > >> Group leader in structural biology of protein-RNA complexes and viral > >> proteins > >> Joint appointment in EMBL Genome Biology Programme > >> Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host > >> Cell Interactions (UVHCI) > >> ** > >> > >> Email: cus...@embl.fr > >> Website: http://www.embl.fr > >> Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 > >> Fax:(33) 4 76 20 7199 > >> Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, > >> 38042 Grenoble Cedex 9, France > >> Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, > >> 6 Rue Jules Horowitz, 38042 Grenoble, F
Re: [ccp4bb] twinning problem ?
Dear Stephen, I have seen a similar effect in the structure of F1-ATPase complexed with the full length inhibitor protein. The inhibitor is a dimer, and it actually couples 2 copies of the ATPase, but it crystallised with only one copy of the ATPase per asymmetric unit. When I solved the structure by MR, I saw additional density that could not be accounted for. The extra density was, in fact, a second ATPase molecule that was related to the first by a 120 degree rotation about the pseudo 3-fold axis of the enzyme. The "dimers" were packing with statistical disorder in the crystal lattice. This gave rise to clear streaking between Bragg spots in the diffraction images in a direction that was consistent with that expected from the statistical packing of the inhibitor linked dimers. Two copies of F1 were included in the refinement, each with occupancy 0.5. the final Rfree was 27.7% (2.8A data). Prior to introduction of the second copy of F1, the Rfree was 37%. More details are in Cabezon et al., NSMB 10, 744-750, 2003 Best wishes, Andrew On 11 Mar 2014, at 14:04, Stephen Cusack wrote: > Dear All, >I have 2.6 A data and unambiguous molecular replacement solution for two > copies/asymmetric unit of a 80 K protein for a crystal integrated > in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. > Refinement allowed rebuilding/completion of the model in the noraml way but > the R-free does not go below 30%. The map in the model regions looks > generally fine but there is a lot > of extra positive density in the solvent regions (some of it looking like > weak density for helices and strands) and unexpected positive peaks within > the model region. > Careful inspection allowed manual positioning of a completely different, > overlapping solution for the dimer which fits the extra density perfectly. > The two incompatible solutions are related by a 2-fold axis parallel to a. > This clearly suggests some kind of twinning. However twinning analysis > programmes (e.g. Phenix-Xtriage), while suggesting the potentiality > of pseudo-merohedral twinning (-h, l, k) do not reveal > any significant twinning fraction and proclaim the data likely to be > untwinned. (NB. The programmes do however highlight a > non-crystallographic translation and there are systematic intensity > differences in the data). Refinement, including this twinning law made no > difference > since the estimated twinning fraction was 0.02. Yet the extra density is > clearly there and I know exactly the real-space transformation between the > two packing solutions. > How can I best take into account this alternative solution (occupancy seems > to be around 20-30%) in the refinement ? > thanks for your suggestions > Stephen > > -- > > ** > Dr. Stephen Cusack, > Head of Grenoble Outstation of EMBL > Group leader in structural biology of protein-RNA complexes and viral proteins > Joint appointment in EMBL Genome Biology Programme > Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell > Interactions (UVHCI) > ** > > Email:cus...@embl.fr > Website: http://www.embl.fr > Tel: (33) 4 76 20 7238Secretary (33) 4 76 20 7123 > > Fax:(33) 4 76 20 7199 > Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, > 38042 Grenoble Cedex 9, France > Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, > 6 Rue Jules Horowitz, 38042 Grenoble, France > **
Re: [ccp4bb] twinning problem ?
Not sure I understand why having statistical disorder makes for streaks--does the crystal then have a whole range of unit cell constants, with the spot at the most prevalent value, and the streaks are the "tails" of the distribution? If so, doesn't having the streak imply a really wide range of constants? And how would this be different from mosaicity? My guess is that this is not the right picture, and this is indeed roughly what mosaicity is. Alternatively, perhaps the streaks are interpreted as the result of a duality between the "unit cell," which yields spots, and a "super cell" which is so large that it yields extremely close "spots" which are indistinguishable from lines/streaks. Usually this potential super cell is squelched by destructive interference due to each component unit cell being very nearly identical, but here the destructive interference doesn't happen because each component unit cell differs quite a bit from its fellows. And I guess in the latter case the "supercell" would have its cell constant (in the direction of the streaks) equal to (or a function of) the coherence length of the incident radiation? I know some attempts have been (successfully) made to use diffuse scattering, but has anyone used the streak intensities to determine interesting features of the crystallized protein? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew Leslie Sent: Wednesday, March 12, 2014 12:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] twinning problem ? Dear Stephen, I have seen a similar effect in the structure of F1-ATPase complexed with the full length inhibitor protein. The inhibitor is a dimer, and it actually couples 2 copies of the ATPase, but it crystallised with only one copy of the ATPase per asymmetric unit. When I solved the structure by MR, I saw additional density that could not be accounted for. The extra density was, in fact, a second ATPase molecule that was related to the first by a 120 degree rotation about the pseudo 3-fold axis of the enzyme. The "dimers" were packing with statistical disorder in the crystal lattice. This gave rise to clear streaking between Bragg spots in the diffraction images in a direction that was consistent with that expected from the statistical packing of the inhibitor linked dimers. Two copies of F1 were included in the refinement, each with occupancy 0.5. the final Rfree was 27.7% (2.8A data). Prior to introduction of the second copy of F1, the Rfree was 37%. More details are in Cabezon et al., NSMB 10, 744-750, 2003 Best wishes, Andrew On 11 Mar 2014, at 14:04, Stephen Cusack wrote: > Dear All, >I have 2.6 A data and unambiguous molecular replacement solution > for two copies/asymmetric unit of a 80 K protein for a crystal integrated in > P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. > Refinement allowed rebuilding/completion of the model in the noraml > way but the R-free does not go below 30%. The map in the model regions looks > generally fine but there is a lot of extra positive density in the solvent > regions (some of it looking like weak density for helices and strands) and > unexpected positive peaks within the model region. > Careful inspection allowed manual positioning of a completely different, > overlapping solution for the dimer which fits the extra density perfectly. > The two incompatible solutions are related by a 2-fold axis parallel to a. > This clearly suggests some kind of twinning. However twinning analysis > programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of > pseudo-merohedral twinning (-h, l, k) do not reveal any significant > twinning fraction and proclaim the data likely to be untwinned. (NB. > The programmes do however highlight a non-crystallographic translation and > there are systematic intensity differences in the data). Refinement, > including this twinning law made no difference since the estimated twinning > fraction was 0.02. Yet the extra density is clearly there and I know exactly > the real-space transformation between the two packing solutions. > How can I best take into account this alternative solution (occupancy seems > to be around 20-30%) in the refinement ? > thanks for your suggestions > Stephen > > -- > > ** > Dr. Stephen Cusack, > Head of Grenoble Outstation of EMBL > Group leader in structural biology of protein-RNA complexes and viral > proteins Joint appointment in EMBL Genome Biology Programme Director > of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell > Interactions (UVHCI) > **
Re: [ccp4bb] twinning problem ?
ter case the "supercell" would have its cell > constant (in the direction of the streaks) equal to (or a function of) the > coherence length of the incident radiation? > > I know some attempts have been (successfully) made to use diffuse > scattering, but has anyone used the streak intensities to determine > interesting features of the crystallized protein? > > JPK > > > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Andrew Leslie > Sent: Wednesday, March 12, 2014 12:25 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] twinning problem ? > > Dear Stephen, > > I have seen a similar effect in the structure of > F1-ATPase complexed with the full length inhibitor > protein. The inhibitor is a dimer, and it actually > couples 2 copies of the ATPase, but it > crystallised with only one copy of the ATPase per > asymmetric unit. When I solved the structure by > MR, I saw additional density that could not be > accounted for. The extra density was, in fact, a > second ATPase molecule that was related to the > first by a 120 degree rotation about the pseudo > 3-fold axis of the enzyme. The "dimers" were > packing with statistical disorder in the crystal > lattice. This gave rise to clear streaking between > Bragg spots in the diffraction images in a > direction that was consistent with that expected > from the statistical packing of the inhibitor > linked dimers. > > Two copies of F1 were included in the refinement, each with occupancy 0.5. > the final Rfree was 27.7% (2.8A data). Prior to introduction of the second > copy of F1, the Rfree was 37%. > > More details are in Cabezon et al., NSMB 10, 744-750, 2003 > > Best wishes, > > Andrew > > > > On 11 Mar 2014, at 14:04, Stephen Cusack wrote: > >> Dear All, >>I have 2.6 A data and unambiguous molecular replacement solution >> for two copies/asymmetric unit of a 80 K protein for a crystal >> integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, >> c=138.9. >> Refinement allowed rebuilding/completion of the model in the noraml >> way but the R-free does not go below 30%. The map in the model regions >> looks generally fine but there is a lot of extra positive density in >> the solvent regions (some of it looking like weak density for helices >> and strands) and unexpected positive peaks within the model region. >> Careful inspection allowed manual positioning of a completely different, >> overlapping solution for the dimer which fits the extra density >> perfectly. >> The two incompatible solutions are related by a 2-fold axis parallel to >> a. >> This clearly suggests some kind of twinning. However twinning analysis >> programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of >> pseudo-merohedral twinning (-h, l, k) do not reveal any significant >> twinning fraction and proclaim the data likely to be untwinned. (NB. >> The programmes do however highlight a non-crystallographic translation >> and there are systematic intensity differences in the data). Refinement, >> including this twinning law made no difference since the estimated >> twinning fraction was 0.02. Yet the extra density is clearly there and I >> know exactly the real-space transformation between the two packing >> solutions. >> How can I best take into account this alternative solution (occupancy >> seems to be around 20-30%) in the refinement ? >> thanks for your suggestions >> Stephen >> >> -- >> >> ** >> Dr. Stephen Cusack, >> Head of Grenoble Outstation of EMBL >> Group leader in structural biology of protein-RNA complexes and viral >> proteins Joint appointment in EMBL Genome Biology Programme Director >> of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell >> Interactions (UVHCI) >> ** >> >> Email: cus...@embl.fr >> Website: http://www.embl.fr >> Tel: (33) 4 76 20 7238Secretary (33) 4 76 20 7123 >> Fax:(33) 4 76 20 7199 >> Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, >> 38042 Grenoble Cedex 9, France >> Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, >> 6 Rue Jules Horowitz, 38042 Grenoble, France >> ** > 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] twinning problem ?
Dear Jacob For a review of this topic see http://www.tandfonline.com/doi/full/10.1080/08893110310001643551#.UyCVLikgGc0 I also refer you to the more recent OUP IUCr book Chayen, Helliwell and Snell ie which includes these topics:- http://global.oup.com/academic/product/macromolecular-crystallization-and-crystal-perfection-9780199213252;jsessionid=5564F908743CCE57BAD506586B47B6CC?cc=gb&lang=en&; I declare a 'perceived conflict of interest' in making this book suggestion to you. Best wishes John Prof John R Helliwell DSc > On 12 Mar 2014, at 16:59, "Keller, Jacob" wrote: > > Not sure I understand why having statistical disorder makes for streaks--does > the crystal then have a whole range of unit cell constants, with the spot at > the most prevalent value, and the streaks are the "tails" of the > distribution? If so, doesn't having the streak imply a really wide range of > constants? And how would this be different from mosaicity? My guess is that > this is not the right picture, and this is indeed roughly what mosaicity is. > > Alternatively, perhaps the streaks are interpreted as the result of a duality > between the "unit cell," which yields spots, and a "super cell" which is so > large that it yields extremely close "spots" which are indistinguishable from > lines/streaks. Usually this potential super cell is squelched by destructive > interference due to each component unit cell being very nearly identical, but > here the destructive interference doesn't happen because each component unit > cell differs quite a bit from its fellows. > > And I guess in the latter case the "supercell" would have its cell constant > (in the direction of the streaks) equal to (or a function of) the coherence > length of the incident radiation? > > I know some attempts have been (successfully) made to use diffuse scattering, > but has anyone used the streak intensities to determine interesting features > of the crystallized protein? > > JPK > > > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew > Leslie > Sent: Wednesday, March 12, 2014 12:25 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] twinning problem ? > > Dear Stephen, > > I have seen a similar effect in the structure of > F1-ATPase complexed with the full length inhibitor protein. The inhibitor is > a dimer, and it actually couples 2 copies of the ATPase, but it crystallised > with only one copy of the ATPase per asymmetric unit. When I solved the > structure by MR, I saw additional density that could not be accounted for. > The extra density was, in fact, a second ATPase molecule that was related to > the first by a 120 degree rotation about the pseudo 3-fold axis of the > enzyme. The "dimers" were packing with statistical disorder in the crystal > lattice. This gave rise to clear streaking between Bragg spots in the > diffraction images in a direction that was consistent with that expected from > the statistical packing of the inhibitor linked dimers. > > Two copies of F1 were included in the refinement, each with occupancy 0.5. > the final Rfree was 27.7% (2.8A data). Prior to introduction of the second > copy of F1, the Rfree was 37%. > > More details are in Cabezon et al., NSMB 10, 744-750, 2003 > > Best wishes, > > Andrew > > > >> On 11 Mar 2014, at 14:04, Stephen Cusack wrote: >> >> Dear All, >> I have 2.6 A data and unambiguous molecular replacement solution >> for two copies/asymmetric unit of a 80 K protein for a crystal integrated in >> P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. >> Refinement allowed rebuilding/completion of the model in the noraml >> way but the R-free does not go below 30%. The map in the model regions looks >> generally fine but there is a lot of extra positive density in the solvent >> regions (some of it looking like weak density for helices and strands) and >> unexpected positive peaks within the model region. >> Careful inspection allowed manual positioning of a completely different, >> overlapping solution for the dimer which fits the extra density perfectly. >> The two incompatible solutions are related by a 2-fold axis parallel to a. >> This clearly suggests some kind of twinning. However twinning analysis >> programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of >> pseudo-merohedral twinning (-h, l, k) do not reveal any significant >> twinning fraction and proclaim the data likely to be untwinned. (NB. >> The programmes do however highlight a non
Re: [ccp4bb] twinning problem ?
>For any sample, crystalline or not, a generally valid description of >diffraction intensity is it being a Fourier transform of electron density >autocorrelation function. I thought for non-crystalline samples diffraction intensity is simply the Fourier transform of the electron density, not its autocorrelation function. Is that wrong? Anyway, regarding spot streaking, perhaps there is a different, simpler formulation for how they arise, based on the two phenomena: (1) Crystal lattice convoluted with periodic contents, e.g., protein structure in exactly the same orientation (2) Crystal lattice convoluted with aperiodic contents, e.g. n different conformations of a protein loop, randomly sprinkled in the lattice. Option (1) makes normal spots. If there is a lot of scattering material doing (2), then streaks arise due to many "super-cells" occurring, each with an integral number of unit cells, and following a Poisson distribution with regard to frequency according to the number of distinct conformations. Anyway, I thought of this because it might be related to scattering from aperiodic crystals, in which there is no concept of unit cell as far as I know (just frequent distances), which makes them really interesting for thinking about diffraction. See the images here of an aperiodic lattice and its Fourier transform, if interested: http://postimg.org/gallery/1fowdm00/ >Mosaicity is a very different phenomenon. It describes a range of angular >alignments of microcrystals with the same unit cell within the sample. It >broadens diffraction peaks by the same angle irrespective of the data >resolution, but it cannot change the length of diffraction vector for each >Bragg reflection. For this reason, the elongation of the spot on the detector >resulting from mosaicity will be always perpendicular to the diffraction >vector. This is distinct from the statistical disorder, where spot elongation >will be aligned with the crystal lattice and not the detector plane. I have been convinced by some elegant, carefully-thought-out papers that this "microcrystal" conception of the data-processing constant "mosaicity" is basically wrong, and that the primary factor responsible for observed mosaicity is discrepancies in unit cell constants, and not the "microcrystal" picture. I think maybe you are referring here to theoretical mosaicity and not the fitting parameter, so I am not contradicting you. I have seen recently an EM study of protein microcrystals which seems to show actual tilted mosaic domains just as you describe, and can find the reference if desired. >Presence of multiple, similar unit cells in the sample is completely different >and unrelated condition to statistical disorder. Agreed! Jacob
Re: [ccp4bb] twinning problem ?
On 03/12/2014 04:15 PM, Keller, Jacob wrote: For any sample, crystalline or not, a generally valid description of diffraction intensity is it being a Fourier transform of electron density autocorrelation function. I thought for non-crystalline samples diffraction intensity is simply the Fourier transform of the electron density, not its autocorrelation function. Is that wrong? The Fourier transform of electron density is a complex scattering amplitude that by the axiom of quantum mechanics is not a measurable quantity. What is measurable is the module squared of it. In crystallography, it is called either F^2 (formally equal F*Fbar) or somewhat informally diffraction intensity, after one takes into account scaling factors. F*Fbar is the Fourier transform of an electron density autocorrelation function regardless if electron density is periodic or not. For periodic electron density the structure factors are described by sum of delta Dirac functions placed on the reciprocal lattice. These delta functions are multiplied by values of structure factors for corresponding Miller indices. Anyway, regarding spot streaking, perhaps there is a different, simpler formulation for how they arise, based on the two phenomena: (1) Crystal lattice convoluted with periodic contents, e.g., protein structure in exactly the same orientation (2) Crystal lattice convoluted with aperiodic contents, e.g. n different conformations of a protein loop, randomly sprinkled in the lattice. Option (1) makes normal spots. If there is a lot of scattering material doing (2), then streaks arise due to many "super-cells" occurring, each with an integral number of unit cells, and following a Poisson distribution with regard to frequency according to the number of distinct conformations. Anyway, I thought of this because it might be related to scattering from aperiodic crystals, in which there is no concept of unit cell as far as I know (just frequent distances), which makes them really interesting for thinking about diffraction. This formulation cannot describe aperiodic contents. The convolution of a crystal lattice with any function will result in electron density, which has a perfect crystal symmetry of the same periodicity as the starting crystal lattice. See the images here of an aperiodic lattice and its Fourier transform, if interested: http://postimg.org/gallery/1fowdm00/ This is interesting case of pseudocrystal, however because there is no crystal lattice, it is not relevant to (1) or (2). In any case, pentagonal quasilattices are probably not relevant to macromolecular crystallography. Mosaicity is a very different phenomenon. It describes a range of angular alignments of microcrystals with the same unit cell within the sample. It broadens diffraction peaks by the same angle irrespective of the data resolution, but it cannot change the length of diffraction vector for each Bragg reflection. For this reason, the elongation of the spot on the detector resulting from mosaicity will be always perpendicular to the diffraction vector. This is distinct from the statistical disorder, where spot elongation will be aligned with the crystal lattice and not the detector plane. I have been convinced by some elegant, carefully-thought-out papers that this "microcrystal" conception of the data-processing constant "mosaicity" is basically wrong, and that the primary factor responsible for observed mosaicity is discrepancies in unit cell constants, and not the "microcrystal" picture. I think maybe you are referring here to theoretical mosaicity and not the fitting parameter, so I am not contradicting you. I have seen recently an EM study of protein microcrystals which seems to show actual tilted mosaic domains just as you describe, and can find the reference if desired. This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. Zbyszek Presence of multiple, similar unit cells in the sample is completely different and unrelated condition to statistical disorder. Agreed! Jacob -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
Re: [ccp4bb] twinning problem ?
>The Fourier transform of electron density is a complex scattering amplitude >that by the axiom of quantum mechanics is not a measurable quantity. What is >measurable is the module squared of it. In crystallography, it is called either F^2 (formally equal F*Fbar) or somewhat informally diffraction intensity, after one takes into account scaling factors. F*Fbar is the Fourier transform of an electron density autocorrelation function regardless if electron density is periodic or not. For periodic electron density the structure factors are described by sum of delta Dirac functions placed on the reciprocal lattice. These delta functions are multiplied by values of structure factors for corresponding Miller indices. Okay, I may have been confused--I thought that the Fourier transform was essentially acting like an autocorrelation function (since generally Fourier transforms are similar to autocorrelation functions--not clear on the details right now), and I had thought I had heard stories of days of yore handwritten Fourier series calculations to make electron density maps. You're telling me they had to also back-calculate an autocorrelation function? Times were tough! Maybe someone from that generation can chime in about how they dealt with this? >This is interesting case of pseudocrystal, however because there is no crystal >lattice, it is not relevant to (1) or (2). In any case, pentagonal >quasilattices are probably not relevant to macromolecular crystallography. I tried a few simulations to show what I mean but ran out of time--sorry about that. I think I'll probably just drop this. NB Linus Pauling said more forcefully the same prediction about aperiodic crystals in general not existing, pentagonal or otherwise, but was proven dead wrong by now-Nobel laureate Dan Shechtman. Maybe someone will come across an aperiodic protein crystal, or already has and missed it, and stupefy us all. Someone mentioned to me once seeing personally a ten-fold symmetrical diffraction pattern from a protein crystal, but she dismissed it with exactly the argument that Pauling made, I think that it was a twinned cubic space group. >This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. I was thinking of this paper for example (see last line of abstract). Perhaps other crystals are different from lysozyme, though, as you mention. All the best, Jacob Keller Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):848-53. A description of imperfections in protein crystals. Nave C. Author information Abstract An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improving crystal perfection, defining data-collection requirements and for data-processing procedures. Measurements on crystals of tetragonal lysozyme at room temperature and 100 K were made in order to illustrate how parameters describing the crystal imperfections can be obtained. At 100 K, the dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal. PMID: 9757100 [PubMed - indexed for MEDLINE]
Re: [ccp4bb] twinning problem ?
On 03/12/2014 09:02 PM, Keller, Jacob wrote: The Fourier transform of electron density is a complex scattering amplitude that by the axiom of quantum mechanics is not a measurable quantity. What is measurable is the module squared of it. In crystallography, it is called either F^2 (formally equal F*Fbar) or somewhat informally diffraction intensity, after one takes into account scaling factors. F*Fbar is the Fourier transform of an electron density autocorrelation function regardless if electron density is periodic or not. For periodic electron density the structure factors are described by sum of delta Dirac functions placed on the reciprocal lattice. These delta functions are multiplied by values of structure factors for corresponding Miller indices. Okay, I may have been confused--I thought that the Fourier transform was essentially acting like an autocorrelation function (since generally Fourier transforms are similar to autocorrelation functions--not clear on the details right now), and I had thought I had heard stories of days of yore handwritten Fourier series calculations to make electron density maps. You're telling me they had to also back-calculate an autocorrelation function? Times were tough! Maybe someone from that generation can chime in about how they dealt with this? Even in today’s easy times, the fastest way to calculate autocorrelation function is to calculate Fourier transform of the data, calculate F*Fbar and calculate back Fourier transform of it. This is interesting case of pseudocrystal, however because there is no crystal lattice, it is not relevant to (1) or (2). In any case, pentagonal quasilattices are probably not relevant to macromolecular crystallography. I tried a few simulations to show what I mean but ran out of time--sorry about that. I think I'll probably just drop this. NB Linus Pauling said more forcefully the same prediction about aperiodic crystals in general not existing, pentagonal or otherwise, but was proven dead wrong by now-Nobel laureate Dan Shechtman. Maybe someone will come across an aperiodic protein crystal, or already has and missed it, and stupefy us all. Someone mentioned to me once seeing personally a ten-fold symmetrical diffraction pattern from a protein crystal, but she dismissed it with exactly the argument that Pauling made, I think that it was a twinned cubic space group. Unless you are interested in finding curious objects, what would you do with protein quasicrystal? The practices of macromolecular crystallography is about determining 3-dimensional structure of objects being crystallized. Protein quasicrystal are really unlikely to diffract to high enough resolution, and even ignoring all other practical aspects, like writing programs to solve such a structure, chances of building an atomic model are really slim. This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. The interpretation of the data presented in this article is that variation in unit cell between microcrystals induce their spatial misalignment. The data do not show variation of unit cell within individual microscrystalline domains. Tetragonal lysozyme can adopt quite a few variations of the crystal lattice during cryocooling. Depending on the conditions used, resulting mosaicity can vary from 0.1 degree (even for 1mm size crystal) to over 1. degree. Consequently, measured structure factors from a group of tetragonal lysozyme crystal can be quite reproducible, or not. As a test crystal, it should be handled with care. 1 degree mosaicity is not an impediment to high quality measurements. However, high mosaicity tends to correlate with presence of phase transitions during cryo-cooling. If such transition happen during cryo-cooling, crystals of the same protein, even from the same drop, may vary quite a lot in terms of structure factors. Additionally, even similar values of unit cell parameters are not guarantee of isomorphism between crystals. Zbyszek I was thinking of this paper for example (see last line of abstract). Perhaps other crystals are different from lysozyme, though, as you mention. All the best, Jacob Keller Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):848-53. A description of imperfections in protein crystals. Nave C. Author information Abstract An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improvi
Re: [ccp4bb] twinning problem ?
Dear Jacob, Measurement of the reciprocal space maps at reflections with triple axis diffractometry allows experimental separation of mosaicity and strain (variation in unit cell parameter) effects. See eg Boggon et al 2000 Acta Cryst D56, 868-880 http://dx.doi.org/10.1107/S090744495837 for such studies on protein crystals at NSLS. In terms of diffuse scattering the above effects do get mixed in with molecular disorders correlated over many unit cells, and thus a 'diffuse scattering correction to measured Bragg intensities' is done in the most accurate work.But the above effects are separate from molecular disorders over a few unit cells ie which cause the diffraction streaks between Bragg peaks. Then there are the long range and short range temporal vibrations, optic and acoustic modes, in the crystal A workshop held at ALS on diffuse scattering recently suggests a systematic effort is on hand to analyse diffuse X-ray scattering information in MX data sets for improved descriptions of macromolecular structure and dynamics. Archiving of raw diffraction data images would also assist such important objectives. Best wishes, John Prof John R Helliwell DSc On 12 Mar 2014, at 21:15, "Keller, Jacob" wrote: >> For any sample, crystalline or not, a generally valid description of >> diffraction intensity is it being a Fourier transform of electron density >> autocorrelation function. > > I thought for non-crystalline samples diffraction intensity is simply the > Fourier transform of the electron density, not its autocorrelation function. > Is that wrong? > > > > Anyway, regarding spot streaking, perhaps there is a different, simpler > formulation for how they arise, based on the two phenomena: > > (1) Crystal lattice convoluted with periodic contents, e.g., protein > structure in exactly the same orientation > (2) Crystal lattice convoluted with aperiodic contents, e.g. n different > conformations of a protein loop, randomly sprinkled in the lattice. > > Option (1) makes normal spots. If there is a lot of scattering material doing > (2), then streaks arise due to many "super-cells" occurring, each with an > integral number of unit cells, and following a Poisson distribution with > regard to frequency according to the number of distinct conformations. > Anyway, I thought of this because it might be related to scattering from > aperiodic crystals, in which there is no concept of unit cell as far as I > know (just frequent distances), which makes them really interesting for > thinking about diffraction. > > See the images here of an aperiodic lattice and its Fourier transform, if > interested: > > http://postimg.org/gallery/1fowdm00/ > >> Mosaicity is a very different phenomenon. It describes a range of angular >> alignments of microcrystals with the same unit cell within the sample. It >> broadens diffraction peaks by the same angle irrespective of the data >> resolution, but it cannot change the length of diffraction vector for each >> Bragg reflection. For this reason, the elongation of the spot on the >> detector resulting from mosaicity will be always perpendicular to the >> diffraction vector. This is distinct from the statistical disorder, where >> spot elongation will be aligned with the crystal lattice and not the >> detector plane. > > I have been convinced by some elegant, carefully-thought-out papers that this > "microcrystal" conception of the data-processing constant "mosaicity" is > basically wrong, and that the primary factor responsible for observed > mosaicity is discrepancies in unit cell constants, and not the "microcrystal" > picture. I think maybe you are referring here to theoretical mosaicity and > not the fitting parameter, so I am not contradicting you. I have seen > recently an EM study of protein microcrystals which seems to show actual > tilted mosaic domains just as you describe, and can find the reference if > desired. > >> Presence of multiple, similar unit cells in the sample is completely >> different and unrelated condition to statistical disorder. > > Agreed! > > Jacob
Re: [ccp4bb] twinning problem ?
>Unless you are interested in finding curious objects, what would you do with >protein quasicrystal? The practices of macromolecular crystallography is about >determining 3-dimensional structure of objects being crystallized. Protein >quasicrystal are really unlikely to diffract to high enough resolution, and >even ignoring all other practical aspects, like writing programs to solve such >a structure, chances of building an atomic model are really slim. Right, if crystallography is seen as purely a tool for biology I agree. As for curious objects, I think almost all profound breakthroughs come from unadulterated curiosity and not desire for some practical end. Not sure why a priori this should be so, but just consider your favorite scientific breakthrough and whether the scientist set out to make the discovery or not. Some are, but most are not, I think. Maybe aperiodic protein crystals have some important function in biology somewhere, or have unforeseen materials science properties, analogous to silk or something. >> This is easy to test by analyzing diffraction patterns of individual >> crystals. > In practice, the dominant contribution to angular broadening of > diffraction peaks is angular disorder of microdomains, particularly in > cryo-cooled crystals. > However, exceptions do happen, but these rare situations need to be > handled on case by case basis. >The interpretation of the data presented in this article is that variation in >unit cell between microcrystals induce their spatial misalignment. The data do >not show variation of unit cell within individual microscrystalline domains. >Tetragonal lysozyme can adopt quite a few variations of the crystal lattice >during cryocooling. Depending on the conditions used, resulting mosaicity can >vary from 0.1 degree (even for 1mm size crystal) to over 1. degree. Consequently, measured structure factors from a group of tetragonal lysozyme crystal can be quite reproducible, or not. As a test crystal, it should be handled with care. 1 degree mosaicity is not an impediment to high quality measurements. However, high mosaicity tends to correlate with presence of phase transitions during cryo-cooling. If such transition happen during cryo-cooling, crystals of the same protein, even from the same drop, may vary quite a lot in terms of structure factors. Additionally, even similar values of unit cell parameters are not guarantee of isomorphism between crystals. So I think you are saying that tetragonal lysozyme is an atypical case, and that normally the main contributor to the fitted parameter "mosaicity" is the phenomenon of microdomains shifted slightly in orientation. Maybe we can get the author to repeat the study for the other usual-suspect protein crystals to find out the truth, but the score currently seems to be 1-0 in favor of cell parameter shifts versus microcrystal orientation... JPK
Re: [ccp4bb] twinning problem ?
On 03/13/2014 10:55 AM, Keller, Jacob wrote: Unless you are interested in finding curious objects, what would you do with protein quasicrystal? The practices of macromolecular crystallography is about determining 3-dimensional structure of objects being crystallized. Protein quasicrystal are really unlikely to diffract to high enough resolution, and even ignoring all other practical aspects, like writing programs to solve such a structure, chances of building an atomic model are really slim. Right, if crystallography is seen as purely a tool for biology I agree. As for curious objects, I think almost all profound breakthroughs come from unadulterated curiosity and not desire for some practical end. Not sure why a priori this should be so, but just consider your favorite scientific breakthrough and whether the scientist set out to make the discovery or not. Some are, but most are not, I think. Maybe aperiodic protein crystals have some important function in biology somewhere, or have unforeseen materials science properties, analogous to silk or something. This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. The interpretation of the data presented in this article is that variation in unit cell between microcrystals induce their spatial misalignment. The data do not show variation of unit cell within individual microscrystalline domains. Tetragonal lysozyme can adopt quite a few variations of the crystal lattice during cryocooling. Depending on the conditions used, resulting mosaicity can vary from 0.1 degree (even for 1mm size crystal) to over 1. degree. Consequently, measured structure factors from a group of tetragonal lysozyme crystal can be quite reproducible, or not. As a test crystal, it should be handled with care. 1 degree mosaicity is not an impediment to high quality measurements. However, high mosaicity tends to correlate with presence of phase transitions during cryo-cooling. If such transition happen during cryo-cooling, crystals of the same protein, even from the same drop, may vary quite a lot in terms of structure factors. Additionally, even similar values of unit cell parameters are not guarantee of isomorphism between crystals. So I think you are saying that tetragonal lysozyme is an atypical case, and that normally the main contributor to the fitted parameter "mosaicity" is the phenomenon of microdomains shifted slightly in orientation. Maybe we can get the author to repeat the study for the other usual-suspect protein crystals to find out the truth, but the score currently seems to be 1-0 in favor of cell parameter shifts versus microcrystal orientation... No, I claim that the particular crystal studied by Colin Nave (Acta Cryst. 1998, D54: 848) is atypical case. I processed myself hundreds of tetragonal lysozyme data sets acquired on crystals grown and mounted by various people, so I believe that my experience defines better a typical case. The second reference, nicely provided by Colin, does not make the conclusion that "dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal", but rather states that "The analysis further suggests that LT disorder is governed by variability inherent in the cooling process combined with the overall history of the crystal." As you can see on the figure 5A in Juers at al, 2007, the mosaicity is a dominant component of the reflection width for resolution higher than 8A. Only for very low resolutions one can see the effect of unit cell changes. What is important is that the crystal analyzed had a very low mosaicity: less than 0.02 degree before cryo-cooling and less than 0.1 degree after cryo-cooling. The mosacity after cryo-cooling is definitely below typical values. One has to remember that not only unit cell parameters are different for different microdomains, but also their structure factors will vary and can change quite a lot. Cryo-cooled crystals definitely can have high degree of internal non-isomorphism resulting from this effect. Zbyszek -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
Re: [ccp4bb] twinning problem ?
Hi Zbyszek I think this has deviated significantly from twinning problems! I certainly don't claim the 1998 study was typical. The crystal was large by present day standards, no cryoprotectant was used and non uniform drying/cooling rates might have occurred. The Juers et. al. paper includes the statement "However, in most cases [omega] does not dominate, suggesting that [delta]a/a plays a significant role in nearly all of our samples." There is also the Kriminski paper (http://journals.iucr.org/d/issues/2002/03/00/en0056/index.html) which includes the statement " Flash-cooling tetragonal lysozyme crystals degrades diffraction resolution and broadens the distributions of lattice orientations (mosaicity) and lattice spacings. The diffraction resolution strongly correlates with the width of the lattice-spacing distribution." The Diedrichs paper includes "The experience of the author is that for most protein crystals reflections are not markedly elongated along circles corresponding to their d-spacing; therefore, `rotational mosaicity' appears to play a minor role . the model calculations suggest that, apart from inhomogeneity and disorder in unit cells, unit-cell parameter variations are responsible for most of the imperfections that result in poor diffraction properties of crystals. Of course selectively quoting papers can be misleading! Fig. 5A of Juers et al lumps omega and delta a/a together and does not distinguish between the two. The plot is [eta] versus d. The slope of a line fit to this plot gives an estimate of 1/s, while the y intercept estimates [omega] + [delta]a/a. In this case, s is the mosaic block size. To summarise cryocooling can produce a fragmentation in to smaller mosaic blocks with larger angular variation between blocks and a distribution of cell dimensions between blocks and within blocks (elastic strain). It really needs a high resolution diffraction set up (to detect diffracted beam divergences above those given by the incident beam divergence) to distinguish between the various effects. Of course, in some cases, such a set up could reveal certain types of twinning (so I have left the subject of the email unchanged!) Regards Colin -Original Message- From: Zbyszek Otwinowski [mailto:zbys...@work.swmed.edu] Sent: 13 March 2014 21:33 To: ccp4bb Subject: Re: [ccp4bb] twinning problem ? On 03/13/2014 10:55 AM, Keller, Jacob wrote: >> Unless you are interested in finding curious objects, what would you do with >> protein quasicrystal? The practices of macromolecular crystallography is >> about determining 3-dimensional structure of objects being crystallized. >> Protein quasicrystal are really unlikely to diffract to high enough >> resolution, and even ignoring all other practical aspects, like writing >> programs to solve such a structure, chances of building an atomic model are >> really slim. > > Right, if crystallography is seen as purely a tool for biology I agree. As > for curious objects, I think almost all profound breakthroughs come from > unadulterated curiosity and not desire for some practical end. Not sure why a > priori this should be so, but just consider your favorite scientific > breakthrough and whether the scientist set out to make the discovery or not. > Some are, but most are not, I think. Maybe aperiodic protein crystals have > some important function in biology somewhere, or have unforeseen materials > science properties, analogous to silk or something. > >>> This is easy to test by analyzing diffraction patterns of individual >>> crystals. >> In practice, the dominant contribution to angular broadening of >> diffraction peaks is angular disorder of microdomains, particularly in >> cryo-cooled crystals. >> However, exceptions do happen, but these rare situations need to be >> handled on case by case basis. >> The interpretation of the data presented in this article is that variation >> in unit cell between microcrystals induce their spatial misalignment. The >> data do not show variation of unit cell within individual microscrystalline >> domains. >> Tetragonal lysozyme can adopt quite a few variations of the crystal lattice >> during cryocooling. Depending on the conditions used, resulting mosaicity >> can vary from 0.1 degree (even for 1mm size crystal) to over 1. degree. > Consequently, measured structure factors from a group of tetragonal lysozyme > crystal can be quite reproducible, or not. As a test crystal, it should be > handled with care. > 1 degree mosaicity is not an impediment to high quality measurements. > However, high mosaicity tends to correlate with presence of phase transitions > during cryo-cooling. If such transition happen during cryo-cooling, crystals
Re: [ccp4bb] twinning problem ?
At the limit, the microdomain picture leads to powder-diffraction-type spots (rings), provided the block size is relatively large with respect to the unit cell. And as the blocks get smaller, the distinction between "changing unit cell parameters" and "mosaic block misorientation" dissolves. I am wondering, then, what one explains by positing microdomains, actually? Is there strong evidence supporting their existence? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Nave Sent: Thursday, March 13, 2014 7:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] twinning problem ? Hi Zbyszek I think this has deviated significantly from twinning problems! I certainly don't claim the 1998 study was typical. The crystal was large by present day standards, no cryoprotectant was used and non uniform drying/cooling rates might have occurred. The Juers et. al. paper includes the statement "However, in most cases [omega] does not dominate, suggesting that [delta]a/a plays a significant role in nearly all of our samples." There is also the Kriminski paper (http://journals.iucr.org/d/issues/2002/03/00/en0056/index.html) which includes the statement " Flash-cooling tetragonal lysozyme crystals degrades diffraction resolution and broadens the distributions of lattice orientations (mosaicity) and lattice spacings. The diffraction resolution strongly correlates with the width of the lattice-spacing distribution." The Diedrichs paper includes "The experience of the author is that for most protein crystals reflections are not markedly elongated along circles corresponding to their d-spacing; therefore, `rotational mosaicity' appears to play a minor role . the model calculations suggest that, apart from inhomogeneity and disorder in unit cells, unit-cell parameter variations are responsible for most of the imperfections that result in poor diffraction properties of crystals. Of course selectively quoting papers can be misleading! Fig. 5A of Juers et al lumps omega and delta a/a together and does not distinguish between the two. The plot is [eta] versus d. The slope of a line fit to this plot gives an estimate of 1/s, while the y intercept estimates [omega] + [delta]a/a. In this case, s is the mosaic block size. To summarise cryocooling can produce a fragmentation in to smaller mosaic blocks with larger angular variation between blocks and a distribution of cell dimensions between blocks and within blocks (elastic strain). It really needs a high resolution diffraction set up (to detect diffracted beam divergences above those given by the incident beam divergence) to distinguish between the various effects. Of course, in some cases, such a set up could reveal certain types of twinning (so I have left the subject of the email unchanged!) Regards Colin -Original Message- From: Zbyszek Otwinowski [mailto:zbys...@work.swmed.edu] Sent: 13 March 2014 21:33 To: ccp4bb Subject: Re: [ccp4bb] twinning problem ? On 03/13/2014 10:55 AM, Keller, Jacob wrote: >> Unless you are interested in finding curious objects, what would you do with >> protein quasicrystal? The practices of macromolecular crystallography is >> about determining 3-dimensional structure of objects being crystallized. >> Protein quasicrystal are really unlikely to diffract to high enough >> resolution, and even ignoring all other practical aspects, like writing >> programs to solve such a structure, chances of building an atomic model are >> really slim. > > Right, if crystallography is seen as purely a tool for biology I agree. As > for curious objects, I think almost all profound breakthroughs come from > unadulterated curiosity and not desire for some practical end. Not sure why a > priori this should be so, but just consider your favorite scientific > breakthrough and whether the scientist set out to make the discovery or not. > Some are, but most are not, I think. Maybe aperiodic protein crystals have > some important function in biology somewhere, or have unforeseen materials > science properties, analogous to silk or something. > >>> This is easy to test by analyzing diffraction patterns of individual >>> crystals. >> In practice, the dominant contribution to angular broadening of >> diffraction peaks is angular disorder of microdomains, particularly in >> cryo-cooled crystals. >> However, exceptions do happen, but these rare situations need to be >> handled on case by case basis. >> The interpretation of the data presented in this article is that variation >> in unit cell between microcrystals induce their spatial misalignment. The >> data do not show variation of unit cell within individual microscrystalline >> domains. >> Tetr
Re: [ccp4bb] twinning problem ?
Jacob One can have microdomains without a significant increase in misorientation e.g. shift dislocations between domains. However, some misorientation is bound to occur. Not sure I understand your statement " And as the blocks get smaller, the distinction between "changing unit cell parameters" and "mosaic block misorientation" dissolves." There are various topography studies on protein crystals (e.g. Gloria Borgstahl and her collaborators) indicating the presence of microdomains. The effect of microdomains (a mosaic block size parameter) on reflection rotation ranges has even been incorporated in to data processing software. One issue is whether there is a continuous variation of cell dimensions within a domain or domains with different unit cell dimensions. This too can be investigated with a high resolution diffraction set up. Colin PS I think we are both using "domain" and "mosaic block" interchangeably. Let me know if you are making a distinction -Original Message- From: Keller, Jacob [mailto:kell...@janelia.hhmi.org] Sent: 14 March 2014 16:32 To: ccp4bb Subject: Re: [ccp4bb] twinning problem ? At the limit, the microdomain picture leads to powder-diffraction-type spots (rings), provided the block size is relatively large with respect to the unit cell. And as the blocks get smaller, the distinction between "changing unit cell parameters" and "mosaic block misorientation" dissolves. I am wondering, then, what one explains by positing microdomains, actually? Is there strong evidence supporting their existence? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Nave Sent: Thursday, March 13, 2014 7:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] twinning problem ? Hi Zbyszek I think this has deviated significantly from twinning problems! I certainly don't claim the 1998 study was typical. The crystal was large by present day standards, no cryoprotectant was used and non uniform drying/cooling rates might have occurred. The Juers et. al. paper includes the statement "However, in most cases [omega] does not dominate, suggesting that [delta]a/a plays a significant role in nearly all of our samples." There is also the Kriminski paper (http://journals.iucr.org/d/issues/2002/03/00/en0056/index.html) which includes the statement " Flash-cooling tetragonal lysozyme crystals degrades diffraction resolution and broadens the distributions of lattice orientations (mosaicity) and lattice spacings. The diffraction resolution strongly correlates with the width of the lattice-spacing distribution." The Diedrichs paper includes "The experience of the author is that for most protein crystals reflections are not markedly elongated along circles corresponding to their d-spacing; therefore, `rotational mosaicity' appears to play a minor role . the model calculations suggest that, apart from inhomogeneity and disorder in unit cells, unit-cell parameter variations are responsible for most of the imperfections that result in poor diffraction properties of crystals. Of course selectively quoting papers can be misleading! Fig. 5A of Juers et al lumps omega and delta a/a together and does not distinguish between the two. The plot is [eta] versus d. The slope of a line fit to this plot gives an estimate of 1/s, while the y intercept estimates [omega] + [delta]a/a. In this case, s is the mosaic block size. To summarise cryocooling can produce a fragmentation in to smaller mosaic blocks with larger angular variation between blocks and a distribution of cell dimensions between blocks and within blocks (elastic strain). It really needs a high resolution diffraction set up (to detect diffracted beam divergences above those given by the incident beam divergence) to distinguish between the various effects. Of course, in some cases, such a set up could reveal certain types of twinning (so I have left the subject of the email unchanged!) Regards Colin -Original Message- From: Zbyszek Otwinowski [mailto:zbys...@work.swmed.edu] Sent: 13 March 2014 21:33 To: ccp4bb Subject: Re: [ccp4bb] twinning problem ? On 03/13/2014 10:55 AM, Keller, Jacob wrote: >> Unless you are interested in finding curious objects, what would you do with >> protein quasicrystal? The practices of macromolecular crystallography is >> about determining 3-dimensional structure of objects being crystallized. >> Protein quasicrystal are really unlikely to diffract to high enough >> resolution, and even ignoring all other practical aspects, like writing >> programs to solve such a structure, chances of building an atomic model are >> really slim. > > Right, if crystallography is seen as purely a tool for biology I agree. As > for curious objects, I think almost all prof
Re: [ccp4bb] twinning problem ?
>One can have microdomains without a significant increase in misorientation >e.g. shift dislocations between domains. However, some misorientation is bound >to occur. Not sure I understand your statement " And as the blocks get >smaller, the distinction between "changing unit cell parameters" and "mosaic >block misorientation" dissolves." I meant that as the number of unit cells per block decreases, the phenomena arising therefrom and from singly-different unit cells are less and less distinguishable. Further, it becomes arbitrary where one puts the boundaries of one's blocks--in the same crystal, one could draw boundaries to minimizes differences within blocks or to minimize statistical differences between blocks. And the unit cells at the edge of blocks--what does one do with them? Jacob
Re: [ccp4bb] Twinning problem
get the twin law and either refine with phenix.refine twin_law="-h,-k,l" or whatever it suggests, or just add into your Refmac script the line TWIN and it will figure out the twin law for you. You can also detwin data but then you might be throwing away a lot of data. We've now had two cases with twin fractions close to 49% and they can definitely not be refined in a higher symmetry space group. One was P21 the other I222. Jürgen On Mar 26, 2013, at 10:45 AM, Liang Zhang wrote: Hi, All, I got a set of P2(or P21) data for MR. However, the Phenix-Xtriage indicated that it could be a pseudo-merohedral twinning. Does anyone know how to deal with such kind of twinning problem? Thanks. Best, Liang .. 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] Twinning problem
Hello, I would suggest to use several tools (in addition to Phenix's) - CCP4's detwin, the plots generated by truncate before detwinning, the Yeates twinning server and there might be others - to get a good idea of what the twinning fraction is. Here we've had success using CCP4's detwin to "detwin" diffraction data. The resulting mtz file is not equivalent to an mtz file containing data recorded from an untwinned crystal - this detwinning operation is not a perfectly accurate operation... In our case we used the estimate of the twinning fraction obtained from Phenix (which was lower). HTH, Fred. On 26/03/13 15:45, Liang Zhang wrote: Hi, All, I got a set of P2(or P21) data for MR. However, the Phenix-Xtriage indicated that it could be a pseudo-merohedral twinning. Does anyone know how to deal with such kind of twinning problem? Thanks. Best, Liang -- Fred. Vellieux (B.Sc., Ph.D., hdr) ouvrier de la recherche IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] Twinning problem
Hello everyone sorry for the intervention with some basic questions regarding twinning In continuation with the discussion with Liang i would like to ask a question which i faced..i have also solved a structure and the statistics depending on twin laws as described through xtriage, phenix is as follows: operator k,h,-l type pseudomerohedral brotton alpha 0.019 h alpha 0.023 m alpha 0.22 it seems the probable twin fraction in my case is 0.2, now the question is does it mean that in another twin domain ie. twin operator h,k,l the twin fraction will be 0.8 ? On Tue, Mar 26, 2013 at 9:07 PM, vellieux wrote: > Hello, > > I would suggest to use several tools (in addition to Phenix's) - CCP4's > detwin, the plots generated by truncate before detwinning, the Yeates > twinning server and there might be others - to get a good idea of what the > twinning fraction is. > > Here we've had success using CCP4's detwin to "detwin" diffraction data. > The resulting mtz file is not equivalent to an mtz file containing data > recorded from an untwinned crystal - this detwinning operation is not a > perfectly accurate operation... In our case we used the estimate of the > twinning fraction obtained from Phenix (which was lower). > > HTH, > > Fred. > > > On 26/03/13 15:45, Liang Zhang wrote: > > Hi, All, > > I got a set of P2(or P21) data for MR. However, the Phenix-Xtriage > indicated that it could be a pseudo-merohedral twinning. Does anyone know > how to deal with such kind of twinning problem? Thanks. > > Best, > > Liang > > > > -- > Fred. Vellieux (B.Sc., Ph.D., hdr) > ouvrier de la recherche > IBS / ELMA > 41 rue Jules Horowitz > F-38027 Grenoble Cedex 01 > Tel: +33 438789605 > Fax: +33 438785494 > > -- Regards Faisal School of Life Sciences JNU
