Re: [ccp4bb] Help with pseudosymmetry problem
Hi Peter, Can you try to run xtriage and see what it tells you in terms of possible twin laws and merging statistics in higher symmetry space groups? The log file is attached. xtriage does not find any clear signs of pseudosymmetry or higher metric symmetry, but it does detect the pseudo-translation peak at (0.129, 0.475, 0.218) with 10% of the origin height, which it doesn't consider as significant (I get 20% when I do it using FFT) If for some reason no twin laws are found, you can manually change the unit cell on the command line to have beta exactly equal to 90... I didn't do this, as possible twin law was found. Or have I misunderstood the logic? Let me know what the logfile tells you. If the translation is 'special' xtraige will tell you what the approximate pseudo symmetry would be. It doesn't seem to think so. Derek P.S. Great program by the way! xtriage.log Description: Binary data 2008/4/23, Derek Logan [EMAIL PROTECTED]: Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_i Isym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine-rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] Help with pseudosymmetry problem
Frankly when faced with these problems of generating symmetry equivalents i revert to almn, where a) I can guarantee the orthogonalisation is as I expect, and b) it generates an exhaustive set of symmetry equivalent peaks. But that is old technology.. If you have two dimers in the asymmetric unit, and only one self rotation peak, it is reasonable to assume the 2 fold NCS axes are in the same orientation. That doesnt necessarily mean the dimers have to be in exactly the same orientation about that 2 fold, s0 I guess you might expect a split peak for a pseudo translation. another trick that sometimes helps - is there a dimer model with exptl data available? again back to almn but sometimes you can fit the two sets of hklin together. Although theoretically you should get the same information from search with a dimer model matching Fcalc to Fobs. Eleanor Derek Logan wrote: Thanks to everyone who helped with the self RF problem: Eleanor, Ian, Claudine, Pietro Alexei. Eleanor wrote: 1) It is a bit hard to find out how MOLREP defines its orthogonal axes - many programs use X0 || a, Yo || b* and in P21 hence Zortho is || to c* If that is what Molrep does then your 2 fold is in the a c* plane, 21 degrees or 111 degrees from c*. The 2 peaks you see are symmetry equivalents. This was my interpretation. Glad we agree ;-) The documentation says A parallel to X , Cstar parallel to Z As for the Patterson - what height are those peaks relative to the origin? The peaks are u = 0.129, v = 0.473, w = 0.220 (20% of origin peak height) and u = 0.180, v = 0.500, w = 0.248 (19%). What I don't get is why there are two and only one strong 2-fold. 2 dimers in the AU gives 50% solvent, 1 dimer 75%. The crystals diffract to 2.3Å, which would tip the balance in favour of 50% solvent in my opinion. With 2 dimers in the asymm unit and with the non-cryst 2-fold perpendicular to b* you could have such translations between one monomer and another. Would the 2-folds of both dimers have to be very similarly oriented? Maybe one peak masks the other at this resolution? is there a model - easiest to solve it then analyse this sort of stuff later! Believe me, we've been trying for a very long time! The problem is that it's a leucine rich repeat protein with under 30% sequence identity to any of the other LRR models out there. I think the failure of MR is down to a combination of a) the low homology, b) the pseudosymmetry, c) the nature of the LRR, which means you can get MR solutions that are out by one or more repeats. Maybe even the internal symmetry of the whole LRR structure can add to this pathology? We've had some solutions that looked almost right, but we can never see much more than what's already in the MR solution. Ian wrote: The symmetry of the self-RF is explained in detail in the documentation for POLARRFN, in fact I would advise you to use this because you can then plot monoclinic space groups with the unique b axis along the orthogonal Z axis (NCODE = 3) and then the symmetry is *much* easier to interpret. The reason I started using Molrep was that POLARRFN always used to choke on these data. However that problem seems to have disappeared. Using ORTH 3 indeed gives a more interpretable plot, as you say. According to polarrfn.doc the symmetry generated by a 2-fold along b parallel to Z is (180-theta, 180-phi, kappa) so the peak in the list (159,180,180) is the same as (21,0,180) which is a NCS 2-fold that you can see just below centre. The peak (111,0,180) is thus the same as (69,180,180) near the top which is another NCS 2-fold perp to the first generated by the crystallographic 2-fold. Indeed, I see the peak (69, 180, 180) but I don't find it in the list in the log file from Molrep. I thought that list was supposed to be exhaustive. Also the plot is not well documented for Molrep. I wrote to the BB a while ago to ask what the contour levels were but no-one answered. By Googling I found a crystallisation paper where it was described as from 0.5 sigma in steps of 0.5 sigma but that information appears to have come by word of mouth. Also, is it just the north hemisphere, as Claudine put it, that is plotted? Anyway, I feel somewhat wiser now... Derek
Re: [ccp4bb] Help with pseudosymmetry problem
we have NCS rotation (158.56, 180, 0) - rotation matrix [R] and we have two CS operators (P21) - rotation matrix (0 0 0) [1] and (90 90 180) [2]. So, all symmetry related (for [R]) rotations are [1][R][1] = [R] - (158.56, 180, 0) [1][R][2] = [R][2] - ( 111.44 0.0 180.0) [2][R][1] = [2][R] - ( 111.44 0.0 180.0) [2][R][2] - (21.44, 0, 180) Really we have two rotations, because (158.56, 180, 0) and (21.44, 0, 180) are idendical. There is nothing special. If there is pure dimer one rotation is pure 2-fold axis another is 2-fold axis with translation. Alexei On 23 Apr 2008, at 22:29, Derek Logan wrote: Thanks to everyone who helped with the self RF problem: Eleanor, Ian, Claudine, Pietro Alexei. Eleanor wrote: 1) It is a bit hard to find out how MOLREP defines its orthogonal axes - many programs use X0 || a, Yo || b* and in P21 hence Zortho is || to c* If that is what Molrep does then your 2 fold is in the a c* plane, 21 degrees or 111 degrees from c*. The 2 peaks you see are symmetry equivalents. This was my interpretation. Glad we agree ;-) The documentation says A parallel to X , Cstar parallel to Z As for the Patterson - what height are those peaks relative to the origin? The peaks are u = 0.129, v = 0.473, w = 0.220 (20% of origin peak height) and u = 0.180, v = 0.500, w = 0.248 (19%). What I don't get is why there are two and only one strong 2-fold. 2 dimers in the AU gives 50% solvent, 1 dimer 75%. The crystals diffract to 2.3Å, which would tip the balance in favour of 50% solvent in my opinion. With 2 dimers in the asymm unit and with the non-cryst 2-fold perpendicular to b* you could have such translations between one monomer and another. Would the 2-folds of both dimers have to be very similarly oriented? Maybe one peak masks the other at this resolution? is there a model - easiest to solve it then analyse this sort of stuff later! Believe me, we've been trying for a very long time! The problem is that it's a leucine rich repeat protein with under 30% sequence identity to any of the other LRR models out there. I think the failure of MR is down to a combination of a) the low homology, b) the pseudosymmetry, c) the nature of the LRR, which means you can get MR solutions that are out by one or more repeats. Maybe even the internal symmetry of the whole LRR structure can add to this pathology? We've had some solutions that looked almost right, but we can never see much more than what's already in the MR solution. Ian wrote: The symmetry of the self-RF is explained in detail in the documentation for POLARRFN, in fact I would advise you to use this because you can then plot monoclinic space groups with the unique b axis along the orthogonal Z axis (NCODE = 3) and then the symmetry is *much* easier to interpret. The reason I started using Molrep was that POLARRFN always used to choke on these data. However that problem seems to have disappeared. Using ORTH 3 indeed gives a more interpretable plot, as you say. According to polarrfn.doc the symmetry generated by a 2-fold along b parallel to Z is (180-theta, 180-phi, kappa) so the peak in the list (159,180,180) is the same as (21,0,180) which is a NCS 2-fold that you can see just below centre. The peak (111,0,180) is thus the same as (69,180,180) near the top which is another NCS 2-fold perp to the first generated by the crystallographic 2-fold. Indeed, I see the peak (69, 180, 180) but I don't find it in the list in the log file from Molrep. I thought that list was supposed to be exhaustive. Also the plot is not well documented for Molrep. I wrote to the BB a while ago to ask what the contour levels were but no-one answered. By Googling I found a crystallisation paper where it was described as from 0.5 sigma in steps of 0.5 sigma but that information appears to have come by word of mouth. Also, is it just the north hemisphere, as Claudine put it, that is plotted? Anyway, I feel somewhat wiser now... Derek
[ccp4bb] Help with pseudosymmetry problem
Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_iIsym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine- rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek
Re: [ccp4bb] Help with pseudosymmetry problem
Hi Derek, The assymertic unit of the self rotation function is only one hemisphere, for example the north hemisphere. So, if you look on the kappa = 180 °, horizontally in the equator you have the crystallographic axis and 2 perpendicular 2-fold axis on to the other, the one in the near middle is nearly but not really parallel to c*, but sure you will have the symmetric of this 2-fold axis by the crystallogrpahic axis, which is in the list but NOT represented in the stereogramm ... Ok, hope this is not to confusing and that it helps ... cheers claudine Derek Logan wrote: Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_iIsym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine- rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek -- * Dr Claudine MAYER MCF Université Paris 6 LRMA Equipe 12 UMR S 872 Laboratoire de Bactériologie (5ème étage gauche) Faculté de Médecine PITIE-SALPETRIERE 91 bd de l'Hopital 75634 PARIS Cedex 13 tel : 01 40 77 95 56 fax : 01 45 82 75 77 mobil: 06 07 23 51 16 [EMAIL PROTECTED] *
Re: [ccp4bb] Help with pseudosymmetry problem
1) It is a bit hard to find out how MOLREP defines its orthogonal axes - many programs use X0 || a, Yo || b* and in P21 hence Zortho is || to c* If that is what Molrep does then your 2 fold is in the a c* plane, 21 degrees or 111 degrees from c*. The 2 peaks you see are symmetry equivalents. If you draw some slugs related by one of those two folds, then add slugs generated by the crystallography 21 axis, you will see that you always must have a 2 fold at theta and also one at theta + 90 ( Have I been here before?? Yes! ) As for the Patterson - what height are those peaks relative to the origin? With 2 dimers in the asymm unit and with the non-cryst 2-fold perpendicular to b* you could have such translations between one monomer and another. is there a model - easiest to solve it then analyse this sort of stuff later! Eleanor Derek Logan wrote: Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_iIsym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine-rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek
Re: [ccp4bb] Help with pseudosymmetry problem
Peak (21.44, 0, 180) has symmetry-related peak (111.44 0 180). These two peaks are identical because NCS peak (21.44, 0, 180) is pependicular to CS peak (90 90 180) Two perpendicular 2-fold peaks (NCS and CS) generate additional 2-fold axis (111.44 0 180). It's called Klug peak. Regards Alexei On 23 Apr 2008, at 13:39, Derek Logan wrote: Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_iIsym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine-rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek
Re: [ccp4bb] Help with pseudosymmetry problem
Hi Derek The symmetry of the self-RF is explained in detail in the documentation for POLARRFN, in fact I would advise you to use this because you can then plot monoclinic space groups with the unique b axis along the orthogonal Z axis (NCODE = 3) and then the symmetry is *much* easier to interpret. Also POLARRFN allows you to plot all the kappa sections not just 4 selected ones. This can be important if the NCS 2-fold is not exact, i.e. you may see the peaks slightly displaced off the kappa=180 or other point-group sections (60, 90, 120). To do the same with MOLREP requires ~ 70 jobs! Note you may need high resolution data (say 2 Ang or better) for this and also sharpening using ECALC so that the peaks are resolved. According to polarrfn.doc the symmetry generated by a 2-fold along b parallel to Z is (180-theta, 180-phi, kappa) so the peak in the list (159,180,180) is the same as (21,0,180) which is a NCS 2-fold that you can see just below centre. The peak (111,0,180) is thus the same as (69,180,180) near the top which is another NCS 2-fold perp to the first generated by the crystallographic 2-fold. HTH! -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Derek Logan Sent: 23 April 2008 13:40 To: CCP4BB@JISCMAIL.AC.UK Subject: Help with pseudosymmetry problem Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_iIsym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine- rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of
Re: [ccp4bb] Help with pseudosymmetry problem
correction: Peak (21.44, 0, 180) has symmetry-related peak (158.56 180 180). . On 23 Apr 2008, at 13:39, Derek Logan wrote: Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_iIsym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine-rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek
Re: [ccp4bb] Help with pseudosymmetry problem
Thanks to everyone who helped with the self RF problem: Eleanor, Ian, Claudine, Pietro Alexei. Eleanor wrote: 1) It is a bit hard to find out how MOLREP defines its orthogonal axes - many programs use X0 || a, Yo || b* and in P21 hence Zortho is || to c* If that is what Molrep does then your 2 fold is in the a c* plane, 21 degrees or 111 degrees from c*. The 2 peaks you see are symmetry equivalents. This was my interpretation. Glad we agree ;-) The documentation says A parallel to X , Cstar parallel to Z As for the Patterson - what height are those peaks relative to the origin? The peaks are u = 0.129, v = 0.473, w = 0.220 (20% of origin peak height) and u = 0.180, v = 0.500, w = 0.248 (19%). What I don't get is why there are two and only one strong 2-fold. 2 dimers in the AU gives 50% solvent, 1 dimer 75%. The crystals diffract to 2.3Å, which would tip the balance in favour of 50% solvent in my opinion. With 2 dimers in the asymm unit and with the non-cryst 2-fold perpendicular to b* you could have such translations between one monomer and another. Would the 2-folds of both dimers have to be very similarly oriented? Maybe one peak masks the other at this resolution? is there a model - easiest to solve it then analyse this sort of stuff later! Believe me, we've been trying for a very long time! The problem is that it's a leucine rich repeat protein with under 30% sequence identity to any of the other LRR models out there. I think the failure of MR is down to a combination of a) the low homology, b) the pseudosymmetry, c) the nature of the LRR, which means you can get MR solutions that are out by one or more repeats. Maybe even the internal symmetry of the whole LRR structure can add to this pathology? We've had some solutions that looked almost right, but we can never see much more than what's already in the MR solution. Ian wrote: The symmetry of the self-RF is explained in detail in the documentation for POLARRFN, in fact I would advise you to use this because you can then plot monoclinic space groups with the unique b axis along the orthogonal Z axis (NCODE = 3) and then the symmetry is *much* easier to interpret. The reason I started using Molrep was that POLARRFN always used to choke on these data. However that problem seems to have disappeared. Using ORTH 3 indeed gives a more interpretable plot, as you say. According to polarrfn.doc the symmetry generated by a 2-fold along b parallel to Z is (180-theta, 180-phi, kappa) so the peak in the list (159,180,180) is the same as (21,0,180) which is a NCS 2-fold that you can see just below centre. The peak (111,0,180) is thus the same as (69,180,180) near the top which is another NCS 2-fold perp to the first generated by the crystallographic 2-fold. Indeed, I see the peak (69, 180, 180) but I don't find it in the list in the log file from Molrep. I thought that list was supposed to be exhaustive. Also the plot is not well documented for Molrep. I wrote to the BB a while ago to ask what the contour levels were but no-one answered. By Googling I found a crystallisation paper where it was described as from 0.5 sigma in steps of 0.5 sigma but that information appears to have come by word of mouth. Also, is it just the north hemisphere, as Claudine put it, that is plotted? Anyway, I feel somewhat wiser now... Derek
