Re: [ccp4bb] SELF-ROTATION FUNCTION FROM MOLREP
First Q - how good is your data - is there no possibility of twinning or any other distraction? Second Q - To compare those results properly we need to know how the P2 and the P222 cell align - are the cell dimensions more or less the same? But the 2 plots you attach (and the list above) show both very strong 222 symmetry so the most likely assumption is that the pointgroup is P222. The next peak down the list is only 0.13 for one and 0.17 for the other pointgroup, which only borders on significance.. But this doesnt really prove anything - for example, if there is a flexible linker the 2 domains of each molecule may be in different relative orientations . Do you have any MR search model for the 2 domains? I would search with them and see what they predict - on the whole self rotation functions are most comprehensible AFTER the structure is solved! Eleanor On 18 November 2013 09:58, Monica Mittal monica.mitta...@gmail.com wrote: Dear CCp4 users, May anyone help me in interpreting the self-rotation function from molrep. Data can be indexed,scaled equally well in P21 and P222. This protein has two domain linked by a long flexible linker and get cleaved during crystallization. After crystallizing it in many condition, i believed that i found new condition where it may be full length. May anyone please suggest me by looking at the self-rotation function, how many molecule exist in ASU. Matthews coefficient suggest that there would be 2 copy of full length protein or four of truncated protein in P21 space group. For your interpretation, please find attached images of rotation function around K=180 Molrep self rotation peaks are P21 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -0.00 180.000.79 | | 335.78 -0.00 180.000.17 | | 490.00 -170.05 180.000.17 | | 558.71 180.00 180.000.14 | | 6 108.34 180.00 180.000.14 | | 7 117.39 180.00 180.000.13 | | 890.00 90.00 108.540.13 | | 990.00 -90.00 108.540.13 | | 10 144.50 -0.00 180.000.13 | | 1137.57 25.59 179.980.12 | | 12 148.100.00 180.000.12 | | 13 121.736.36 179.620.11 | | 1463.31 -42.27 180.000.11 | | 1590.00 90.00 162.290.11 | | 1690.00 -90.00 162.290.11 | | 1771.12 136.88 180.000.10 | | 1899.17 -180.00 180.000.10 | | 19 144.80 -161.87 180.000.10 | | 2090.00 -138.24 180.000.10 | | 2182.27 -76.45 180.000.10 | | 2290.00 90.00 115.940.10 | | 2390.00 -90.00 115.940.10 | | 24 159.16 27.07 180.000.10 | | 2595.08 -35.92 179.840.10 | | 26 113.86 -139.44 179.540.10 | | 2790.23 -0.00 89.320.10 | | 2820.18 -156.63 179.980.10 | | 29 116.90 -40.40 179.500.10 | | 3063.12 139.60 179.500.10 P222 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -21.41 180.000.13 | | 3 125.320.00 180.000.13 | | 4 151.18 -90.00 180.000.12 | | 590.00 -180.00 90.000.12 | | 6 141.32 26.82 180.000.12 | | 790.00 -42.59 180.000.11 | | 8 127.22 -25.21 180.000.11 | | 937.04 -14.48 180.000.11 | | 1059.17 -129.14 180.000.10 | | 1156.66 -174.09 180.000.09 | | 1240.85 144.82 180.000.09 | | 1354.16 -167.88 180.000.09 | | 14 109.29 -61.09 180.000.09 | | 15 123.12 -84.90 180.000.09 | | 1664.66 -93.79 180.000.09 | | 17 113.09 108.59 179.890.09 | | 1893.84 37.37 179.940.08 | | 1990.00 -33.41 180.000.08 | | 20 131.95 90.00 90.750.08 | | 2153.78 -144.59 180.000.08 | | 2249.15 -43.11 180.000.08 | | 2362.98 -56.60 180.000.08 | | 2461.97 138.06 179.910.08 | | 2596.77 59.47 179.900.08 | | 26 118.74 40.91 179.850.07 | | 27 100.14 57.97 179.860.07 | | 2859.74 -139.98 180.000.07 | | 2958.97 -141.14 180.000.07 | | 30 103.38 59.92 179.810.07 Many Thanks in advance for your kind help. THANK YOU
Re: [ccp4bb] SELF-ROTATION FUNCTION FROM MOLREP
Dear CCP4 Users, In Phenix.xtriage and phaser, the analyses of the Patterson function reveals a significant off-origin peak that is 23.25 % of the origin peak, indicating pseudo translational symmetry at frac. cord. vector 0.0000.5000.022 . It did not indicate any twinng. Data is good with resolution upto 2.8A. The cell dimension in P21 SG is 57.877, 63.321, 108.028, 90, 89.987, 90 and in P222 SG is 57.805, 63.266, 108.053, 90, 90, 90. We can say cell dimension align well in in P21 and P222 SG. For MR template, about 75% of domain1, we have solved in our lab and remaining 25% is available from pdb with 30% sequence identity. It is a transcription factor with ligand bindng domain and DNA binding domain. I tried finding MR solution but does not gave satisfaction result. The Rwork and Rfree stalls around 47% and 53% respectively. Is there any possiblity to guess the no of molecules present in ASU from self-rotation function? If we can then we will be sure of whether it is full length of truncated one. Thank you Monica On 11/18/13, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: First Q - how good is your data - is there no possibility of twinning or any other distraction? Second Q - To compare those results properly we need to know how the P2 and the P222 cell align - are the cell dimensions more or less the same? But the 2 plots you attach (and the list above) show both very strong 222 symmetry so the most likely assumption is that the pointgroup is P222. The next peak down the list is only 0.13 for one and 0.17 for the other pointgroup, which only borders on significance.. But this doesnt really prove anything - for example, if there is a flexible linker the 2 domains of each molecule may be in different relative orientations . Do you have any MR search model for the 2 domains? I would search with them and see what they predict - on the whole self rotation functions are most comprehensible AFTER the structure is solved! Eleanor On 18 November 2013 09:58, Monica Mittal monica.mitta...@gmail.com wrote: Dear CCp4 users, May anyone help me in interpreting the self-rotation function from molrep. Data can be indexed,scaled equally well in P21 and P222. This protein has two domain linked by a long flexible linker and get cleaved during crystallization. After crystallizing it in many condition, i believed that i found new condition where it may be full length. May anyone please suggest me by looking at the self-rotation function, how many molecule exist in ASU. Matthews coefficient suggest that there would be 2 copy of full length protein or four of truncated protein in P21 space group. For your interpretation, please find attached images of rotation function around K=180 Molrep self rotation peaks are P21 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -0.00 180.000.79 | | 335.78 -0.00 180.000.17 | | 490.00 -170.05 180.000.17 | | 558.71 180.00 180.000.14 | | 6 108.34 180.00 180.000.14 | | 7 117.39 180.00 180.000.13 | | 890.00 90.00 108.540.13 | | 990.00 -90.00 108.540.13 | | 10 144.50 -0.00 180.000.13 | | 1137.57 25.59 179.980.12 | | 12 148.100.00 180.000.12 | | 13 121.736.36 179.620.11 | | 1463.31 -42.27 180.000.11 | | 1590.00 90.00 162.290.11 | | 1690.00 -90.00 162.290.11 | | 1771.12 136.88 180.000.10 | | 1899.17 -180.00 180.000.10 | | 19 144.80 -161.87 180.000.10 | | 2090.00 -138.24 180.000.10 | | 2182.27 -76.45 180.000.10 | | 2290.00 90.00 115.940.10 | | 2390.00 -90.00 115.940.10 | | 24 159.16 27.07 180.000.10 | | 2595.08 -35.92 179.840.10 | | 26 113.86 -139.44 179.540.10 | | 2790.23 -0.00 89.320.10 | | 2820.18 -156.63 179.980.10 | | 29 116.90 -40.40 179.500.10 | | 3063.12 139.60 179.500.10 P222 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -21.41 180.000.13 | | 3 125.320.00 180.000.13 | | 4 151.18 -90.00 180.000.12 | | 590.00 -180.00 90.000.12 | | 6 141.32 26.82 180.000.12 | | 790.00 -42.59 180.000.11 | | 8 127.22 -25.21 180.000.11 | | 937.04 -14.48 180.000.11 | | 1059.17 -129.14 180.000.10 | | 1156.66 -174.09 180.000.09
[ccp4bb] AW: [ccp4bb] SELF-ROTATION FUNCTION FROM MOLREP
Dear Monica, strong 222 rotational symmetry plus translational symmetry would give 8 molecules in the unit cell. (4 in the a.u. in P2x and 2 in case of P2x2x2x). (test ALL options!). Do you have models for both the ligand binding domain, or only for the DNA binding domain? You have to search for the domains with separate models, not as a single model with the complete protein since the relative orientation of the domains may differ. I would just run MR jobs for 1, 2 and 3 molecules in the a.u. and for full-length and truncated models and see which one gives best results. If you make a script for it, it is very little work. Good luck! Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Monica Mittal Gesendet: Montag, 18. November 2013 14:37 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] SELF-ROTATION FUNCTION FROM MOLREP Dear CCP4 Users, In Phenix.xtriage and phaser, the analyses of the Patterson function reveals a significant off-origin peak that is 23.25 % of the origin peak, indicating pseudo translational symmetry at frac. cord. vector 0.0000.5000.022 . It did not indicate any twinng. Data is good with resolution upto 2.8A. The cell dimension in P21 SG is 57.877, 63.321, 108.028, 90, 89.987, 90 and in P222 SG is 57.805, 63.266, 108.053, 90, 90, 90. We can say cell dimension align well in in P21 and P222 SG. For MR template, about 75% of domain1, we have solved in our lab and remaining 25% is available from pdb with 30% sequence identity. It is a transcription factor with ligand bindng domain and DNA binding domain. I tried finding MR solution but does not gave satisfaction result. The Rwork and Rfree stalls around 47% and 53% respectively. Is there any possiblity to guess the no of molecules present in ASU from self-rotation function? If we can then we will be sure of whether it is full length of truncated one. Thank you Monica On 11/18/13, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: First Q - how good is your data - is there no possibility of twinning or any other distraction? Second Q - To compare those results properly we need to know how the P2 and the P222 cell align - are the cell dimensions more or less the same? But the 2 plots you attach (and the list above) show both very strong 222 symmetry so the most likely assumption is that the pointgroup is P222. The next peak down the list is only 0.13 for one and 0.17 for the other pointgroup, which only borders on significance.. But this doesnt really prove anything - for example, if there is a flexible linker the 2 domains of each molecule may be in different relative orientations . Do you have any MR search model for the 2 domains? I would search with them and see what they predict - on the whole self rotation functions are most comprehensible AFTER the structure is solved! Eleanor On 18 November 2013 09:58, Monica Mittal monica.mitta...@gmail.com wrote: Dear CCp4 users, May anyone help me in interpreting the self-rotation function from molrep. Data can be indexed,scaled equally well in P21 and P222. This protein has two domain linked by a long flexible linker and get cleaved during crystallization. After crystallizing it in many condition, i believed that i found new condition where it may be full length. May anyone please suggest me by looking at the self-rotation function, how many molecule exist in ASU. Matthews coefficient suggest that there would be 2 copy of full length protein or four of truncated protein in P21 space group. For your interpretation, please find attached images of rotation function around K=180 Molrep self rotation peaks are P21 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -0.00 180.000.79 | | 335.78 -0.00 180.000.17 | | 490.00 -170.05 180.000.17 | | 558.71 180.00 180.000.14 | | 6 108.34 180.00 180.000.14 | | 7 117.39 180.00 180.000.13 | | 890.00 90.00 108.540.13 | | 990.00 -90.00 108.540.13 | | 10 144.50 -0.00 180.000.13 | | 1137.57 25.59 179.980.12 | | 12 148.100.00 180.000.12 | | 13 121.736.36 179.620.11 | | 1463.31 -42.27 180.000.11 | | 1590.00 90.00 162.290.11 | | 1690.00 -90.00 162.290.11 | | 1771.12 136.88 180.000.10 | | 1899.17 -180.00 180.000.10 | | 19 144.80 -161.87 180.000.10 | | 2090.00 -138.24 180.000.10 | | 2182.27 -76.45 180.000.10 | | 2290.00 90.00 115.940.10 | | 2390.00 -90.00 115.940.10 | | 24 159.16
