Re: [ccp4bb] Twinning in C2 ?
Dear Eleanor, many thanks for all your help and suggestions. We finally have solved the mutant structure. The trick was to use the pseudo translation (there was not any 2 fold axis in the self rotation) with MOLREP which found two molecules and then applied the pseudo translation to provide us with the other two molecules yielding a tetramer. I think that initially the programs and also ourselves missed the pseudo translation because it is very close to the crystallographic one (0.49 0 0.48). Many thanks to Peter Zwart who also advised us to follow the same procedure. Demetres Eleanor Dodson wrote: Oh dear - this is tricky! You could have a dimer in the asymmetric unit, or two monomers in the asymm unit which generate dimers using the crystallographic 2 folds. Things worth checking - What does the self rotation show? Is there a clear 2 fold axis which is different from the crystallographic one? If so you can use all of MOLREPs cleverness to use the self rotation definition and the pseudo translation. (Under search parameters you can give the self rotation list of solutions - just use self rotation with 1 or 2 solutions and edit out the top one which will be the crystallographic 2 fold) It will find the pseudo trans vector automatically. If the only 2 fold is the cryst one then you need to search for 2 molecules with the pseudo translation. Eleanor What is the solvent content of the native - the mutant has a smaller volume so less solvent - is that feasible? I think I would generate P1 data Demetres D. Leonidas wrote: Dear Eleanor, Yes the native is a dimer and we did the search using the dimer as a model but we had similar results (i.e. all programs find one molecule). The graphs from TRUNCATE show rather normal and I am attaching a gif file with the plot for the cumulative intensity. As for pseudo-translation running the Analyse Data for MR option in ccp4 in the patterson map we are getting a significant peak at fractional coordinates 0.4897 0.0 0.4767. How this can help ? Do we need to apply this pseudo translation to the solution we are getting from molrep ? many thanks Demetres P.S. I will summarize for the members of the list all the suggestions I will get at the end Eleanor Dodson wrote: You dont say whether the molecules in the native cell form a dimer - if so I would search with that (you may need to turn off the packing search) Or whether there is a pseudo translation vector in the mutant form.. Or what the data analysis graphs from TRUNCATE show - are they normal? Eleanor Demetres D. Leonidas wrote: Dear all, we have encountered a problem in solving one mutant structure. The mutant protein crystallizes in the same space group as the native (C2) but the unit cell dimensions are different. These for the native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant 160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has four molecules in the asymmetric unit while the native had two. When we run molecular replacement all programs (CNS, molrep, and amore) find only two molecules. Phaser finds four but when we try to refine the Rfree does not drop below 0.44 if we use four molecules and 0.53 if we only use two no matter how well we built the molecule and regardless of any addition of water molecules (the resolution of the data is 2.1). The interesting thing is that in the electron density map we can clearly see density for a substrate analog that was included in the crystallization media. Do you thing that we have a case of twinning here ? We have to mention that Tod Yates served did not indicate any perfect merohedral twinning (partial merohedral twinning for this space group is not possible). We would appreciate any comments Many thanks Demetres -- Demetres D. Leonidas, Ph.D. Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr ==
Re: [ccp4bb] Twinning in C2 ?
Dear Eleanor, Yes the native is a dimer and we did the search using the dimer as a model but we had similar results (i.e. all programs find one molecule). The graphs from TRUNCATE show rather normal and I am attaching a gif file with the plot for the cumulative intensity. As for pseudo-translation running the Analyse Data for MR option in ccp4 in the patterson map we are getting a significant peak at fractional coordinates 0.4897 0.0 0.4767. How this can help ? Do we need to apply this pseudo translation to the solution we are getting from molrep ? many thanks Demetres P.S. I will summarize for the members of the list all the suggestions I will get at the end Eleanor Dodson wrote: You dont say whether the molecules in the native cell form a dimer - if so I would search with that (you may need to turn off the packing search) Or whether there is a pseudo translation vector in the mutant form.. Or what the data analysis graphs from TRUNCATE show - are they normal? Eleanor Demetres D. Leonidas wrote: Dear all, we have encountered a problem in solving one mutant structure. The mutant protein crystallizes in the same space group as the native (C2) but the unit cell dimensions are different. These for the native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant 160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has four molecules in the asymmetric unit while the native had two. When we run molecular replacement all programs (CNS, molrep, and amore) find only two molecules. Phaser finds four but when we try to refine the Rfree does not drop below 0.44 if we use four molecules and 0.53 if we only use two no matter how well we built the molecule and regardless of any addition of water molecules (the resolution of the data is 2.1). The interesting thing is that in the electron density map we can clearly see density for a substrate analog that was included in the crystallization media. Do you thing that we have a case of twinning here ? We have to mention that Tod Yates served did not indicate any perfect merohedral twinning (partial merohedral twinning for this space group is not possible). We would appreciate any comments Many thanks Demetres -- Demetres D. Leonidas, Ph.D. Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr == inline: cumulative.gif
Re: [ccp4bb] Twinning in C2 ?
You dont say whether the molecules in the native cell form a dimer - if so I would search with that (you may need to turn off the packing search) Or whether there is a pseudo translation vector in the mutant form.. Or what the data analysis graphs from TRUNCATE show - are they normal? Eleanor Demetres D. Leonidas wrote: Dear all, we have encountered a problem in solving one mutant structure. The mutant protein crystallizes in the same space group as the native (C2) but the unit cell dimensions are different. These for the native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant 160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has four molecules in the asymmetric unit while the native had two. When we run molecular replacement all programs (CNS, molrep, and amore) find only two molecules. Phaser finds four but when we try to refine the Rfree does not drop below 0.44 if we use four molecules and 0.53 if we only use two no matter how well we built the molecule and regardless of any addition of water molecules (the resolution of the data is 2.1). The interesting thing is that in the electron density map we can clearly see density for a substrate analog that was included in the crystallization media. Do you thing that we have a case of twinning here ? We have to mention that Tod Yates served did not indicate any perfect merohedral twinning (partial merohedral twinning for this space group is not possible). We would appreciate any comments Many thanks Demetres
Re: [ccp4bb] Twinning in C2 ?
Hi Demetres, not sure if this is going to be usefull, but here I go. Your native has a twin law that and your cell is pseudo C222. The mutant is not. pseudo merohedral twinning is not handled well by the twin server you derscribe as it relies on lookup tables. There is no obvious 'perfect' relation between two unit cells. The ratio between the two unit cell volumes is about 1.8 and a sublattice of your native cell that comes close to the lattice of your mutant does exist. iotbx.explore_metric_symmetry tries to find you possible relations. It uses niggli settings though and the output is not very user (or even developer) friendly: -- Mutant niggli cell : 32.3 81.8 90.1 76.8 79.7 78.6 Native niggli cell : 33.1 53.2 73.4 90.3 90.0 108.1 / 100 \ matrix : M = | 021 | \ 001 / (matrix M acts on the real space basis vectors of the Native niggli cell) Additional Niggli transform: x-y,-y-z,y Additional similarity transform: x,y,z Resulting unit cell : 33.1 90.5 90.9 67.8 79.5 79.5 Deviations :-2.5 -10.6 -0.8 8.9 0.2 -0.8 Deviations for unit cell lengths are listed in %. Angular deviations are listed in degrees. -- Details of what the matrix M means is found in http://www.ccp4.ac.uk/newsletters/newsletter44/articles/explore_metric_symmetry.html It is too early for me to understand if the pseudo translation you see at (1/2,0,1/2) is related to the transfomration shown above, but if you multiply this translation in C2, you do end up with a smaller unit cell. why don't you try this: run xtriage (a latest version) on your mutant data and see what the patterson analyses tells you what it thinks the unit cell is if the patterson peak were a true crystallographic operator. Take that unit cell and compare it to your native (iotbx.explore_metric_symmetry might be usefull). Again, I am not sure that a relation is there, but if it is real, you can get the coordinates for your mutant structure almost directly from your native structure. The relations one sees can be deceiving though, it could be crystallographic numerology. Cheers Peter 2007/7/13, Demetres D. Leonidas [EMAIL PROTECTED]: Dear all, we have encountered a problem in solving one mutant structure. The mutant protein crystallizes in the same space group as the native (C2) but the unit cell dimensions are different. These for the native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant 160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has four molecules in the asymmetric unit while the native had two. When we run molecular replacement all programs (CNS, molrep, and amore) find only two molecules. Phaser finds four but when we try to refine the Rfree does not drop below 0.44 if we use four molecules and 0.53 if we only use two no matter how well we built the molecule and regardless of any addition of water molecules (the resolution of the data is 2.1). The interesting thing is that in the electron density map we can clearly see density for a substrate analog that was included in the crystallization media. Do you thing that we have a case of twinning here ? We have to mention that Tod Yates served did not indicate any perfect merohedral twinning (partial merohedral twinning for this space group is not possible). We would appreciate any comments Many thanks Demetres -- Demetres D. Leonidas, Ph.D. Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr ==
Re: [ccp4bb] Twinning in C2 ?
Oh dear - this is tricky! You could have a dimer in the asymmetric unit, or two monomers in the asymm unit which generate dimers using the crystallographic 2 folds. Things worth checking - What does the self rotation show? Is there a clear 2 fold axis which is different from the crystallographic one? If so you can use all of MOLREPs cleverness to use the self rotation definition and the pseudo translation. (Under search parameters you can give the self rotation list of solutions - just use self rotation with 1 or 2 solutions and edit out the top one which will be the crystallographic 2 fold) It will find the pseudo trans vector automatically. If the only 2 fold is the cryst one then you need to search for 2 molecules with the pseudo translation. Eleanor What is the solvent content of the native - the mutant has a smaller volume so less solvent - is that feasible? I think I would generate P1 data Demetres D. Leonidas wrote: Dear Eleanor, Yes the native is a dimer and we did the search using the dimer as a model but we had similar results (i.e. all programs find one molecule). The graphs from TRUNCATE show rather normal and I am attaching a gif file with the plot for the cumulative intensity. As for pseudo-translation running the Analyse Data for MR option in ccp4 in the patterson map we are getting a significant peak at fractional coordinates 0.4897 0.0 0.4767. How this can help ? Do we need to apply this pseudo translation to the solution we are getting from molrep ? many thanks Demetres P.S. I will summarize for the members of the list all the suggestions I will get at the end Eleanor Dodson wrote: You dont say whether the molecules in the native cell form a dimer - if so I would search with that (you may need to turn off the packing search) Or whether there is a pseudo translation vector in the mutant form.. Or what the data analysis graphs from TRUNCATE show - are they normal? Eleanor Demetres D. Leonidas wrote: Dear all, we have encountered a problem in solving one mutant structure. The mutant protein crystallizes in the same space group as the native (C2) but the unit cell dimensions are different. These for the native structure are 101.2 33.1 73.4 90 90.3 90 and for the mutant 160.4 32.3 107.0 90 125.7 90. As a result the mutant structure has four molecules in the asymmetric unit while the native had two. When we run molecular replacement all programs (CNS, molrep, and amore) find only two molecules. Phaser finds four but when we try to refine the Rfree does not drop below 0.44 if we use four molecules and 0.53 if we only use two no matter how well we built the molecule and regardless of any addition of water molecules (the resolution of the data is 2.1). The interesting thing is that in the electron density map we can clearly see density for a substrate analog that was included in the crystallization media. Do you thing that we have a case of twinning here ? We have to mention that Tod Yates served did not indicate any perfect merohedral twinning (partial merohedral twinning for this space group is not possible). We would appreciate any comments Many thanks Demetres
