Re: [ccp4bb] Compounded problems for molecular replacement
You don't say whether you have any indication of non cryst translation or likely dimer NC axes? The self rotation can help sometimes to select likely pairings - for instance if one (or both) domain(s) is forming a dimer. Eleanor On 26 Oct 2012, at 13:43, Bosch, Juergen wrote: We had recently a similar case. Indexed in P2x2x2x but truly was P21 with variable amount of twin fraction depending on the dataset beamline between 9% and 40%. We were able to solve it using phenix.ensembler using all available known structures plus our various homology models. We still have not gone back and done a postmortem analysis of why we failed with a highly similar homolog to find a solution in the first place with either Phaser, Molrep or Phenix using single models. It might help to process your data in XDS to take care of the overlaps a bit better in case you have not done so already. Here's a link advertisement on Hain et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22982544 /advertisement off. Jürgen .. 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 On Oct 26, 2012, at 8:27 AM, Seijo, Jose A. Cuesta wrote: Hi all, I am dealing with a molecular replacement problem for a 60KDa protein composed of 2 rigid domains joined by a flexible linker which can move relative to each other. Sequence identity for my best model is 46% evenly spread, so in principle this should be a tractable problem. Then the problems start to pile up: a) The unit cell is 56.7Å, 288.5Å, 69.4Å, 90 93.5, 90. Spacegroup P21. Rmerge 12% to 2.4Å. The data also merges relatively well (Rmerge 16%) in P222 with the same a, c and b axes, now of course in that order. In the P21 case, that corresponds to 4 monomers in the asymmetric unit with a solvent content of approx. 50%, giving me 8 domains to find if I separate them. b) The 288 axis means that my data show some overlap in almost all orientations (might be corrected in the future with new datasets), so that my low resolution data are likely unreliable. c) Intensity distributions suggest twinning in either point groups. Actually, they are beyond the perfect twinning case, which I attribute to the reflection overlaps making the strong reflections weaker (integration box too small) and the small stronger (from tails of adjacent strong ones). Of course the latest would mean that the twin fraction estimation is unreliable, but all moments, etc show perfect twin statistics, so I am assuming that there is indeed perfect twinning of some sort. So, the question is, what is the best strategy to deal with this many (4 or 8) body / noisy / twinned problem? I am trying EPMR with many bodies, but I suspect the twinning would throw it out of the right track, and one domain seems to be too little of the diffracting matter to show any sort of discriminations between solutions and non-solutions if do the usual serial searches. I plan to let autotracing programs be the judge of success, but I am not sure of how well those can deal with twinning. Can Arp-Warp use twinned data? Thanks in advance for any tips. Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk
[ccp4bb] Compounded problems for molecular replacement
Hi all, I am dealing with a molecular replacement problem for a 60KDa protein composed of 2 rigid domains joined by a flexible linker which can move relative to each other. Sequence identity for my best model is 46% evenly spread, so in principle this should be a tractable problem. Then the problems start to pile up: a) The unit cell is 56.7Å, 288.5Å, 69.4Å, 90 93.5, 90. Spacegroup P21. Rmerge 12% to 2.4Å. The data also merges relatively well (Rmerge 16%) in P222 with the same a, c and b axes, now of course in that order. In the P21 case, that corresponds to 4 monomers in the asymmetric unit with a solvent content of approx. 50%, giving me 8 domains to find if I separate them. b) The 288 axis means that my data show some overlap in almost all orientations (might be corrected in the future with new datasets), so that my low resolution data are likely unreliable. c) Intensity distributions suggest twinning in either point groups. Actually, they are beyond the perfect twinning case, which I attribute to the reflection overlaps making the strong reflections weaker (integration box too small) and the small stronger (from tails of adjacent strong ones). Of course the latest would mean that the twin fraction estimation is unreliable, but all moments, etc show perfect twin statistics, so I am assuming that there is indeed perfect twinning of some sort. So, the question is, what is the best strategy to deal with this many (4 or 8) body / noisy / twinned problem? I am trying EPMR with many bodies, but I suspect the twinning would throw it out of the right track, and one domain seems to be too little of the diffracting matter to show any sort of discriminations between solutions and non-solutions if do the usual serial searches. I plan to let autotracing programs be the judge of success, but I am not sure of how well those can deal with twinning. Can Arp-Warp use twinned data? Thanks in advance for any tips. Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk
Re: [ccp4bb] Compounded problems for molecular replacement
We had recently a similar case. Indexed in P2x2x2x but truly was P21 with variable amount of twin fraction depending on the dataset beamline between 9% and 40%. We were able to solve it using phenix.ensembler using all available known structures plus our various homology models. We still have not gone back and done a postmortem analysis of why we failed with a highly similar homolog to find a solution in the first place with either Phaser, Molrep or Phenix using single models. It might help to process your data in XDS to take care of the overlaps a bit better in case you have not done so already. Here's a link advertisement on Hain et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22982544 /advertisement off. Jürgen .. 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 On Oct 26, 2012, at 8:27 AM, Seijo, Jose A. Cuesta wrote: Hi all, I am dealing with a molecular replacement problem for a 60KDa protein composed of 2 rigid domains joined by a flexible linker which can move relative to each other. Sequence identity for my best model is 46% evenly spread, so in principle this should be a tractable problem. Then the problems start to pile up: a) The unit cell is 56.7Å, 288.5Å, 69.4Å, 90 93.5, 90. Spacegroup P21. Rmerge 12% to 2.4Å. The data also merges relatively well (Rmerge 16%) in P222 with the same a, c and b axes, now of course in that order. In the P21 case, that corresponds to 4 monomers in the asymmetric unit with a solvent content of approx. 50%, giving me 8 domains to find if I separate them. b) The 288 axis means that my data show some overlap in almost all orientations (might be corrected in the future with new datasets), so that my low resolution data are likely unreliable. c) Intensity distributions suggest twinning in either point groups. Actually, they are beyond the perfect twinning case, which I attribute to the reflection overlaps making the strong reflections weaker (integration box too small) and the small stronger (from tails of adjacent strong ones). Of course the latest would mean that the twin fraction estimation is unreliable, but all moments, etc show perfect twin statistics, so I am assuming that there is indeed perfect twinning of some sort. So, the question is, what is the best strategy to deal with this many (4 or 8) body / noisy / twinned problem? I am trying EPMR with many bodies, but I suspect the twinning would throw it out of the right track, and one domain seems to be too little of the diffracting matter to show any sort of discriminations between solutions and non-solutions if do the usual serial searches. I plan to let autotracing programs be the judge of success, but I am not sure of how well those can deal with twinning. Can Arp-Warp use twinned data? Thanks in advance for any tips. Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dkmailto:josea.cuesta.se...@carlsberglab.dk
Re: [ccp4bb] Compounded problems for molecular replacement
I had a similar issue (8 chains in a large ASU, 2.4 A resolution, 29% homology, possibly--and ultimately confirmed--twinned data). Here is how I solved my structure (not pretty!): 1. Phaser was used to find an initial placement for the chains using (what turned out to be) the twin maps. This actually worked fine to get a grossly correct position for all chains in the ASU. But the low homology structures were pretty much useless as a starting point for refinement in Refmac, and the twin maps were, um, just bad, as you would expect. I used this structure as a starting point for phases for the next step. 2. Once I had that, is used Parrot and Buccaneer to do density modification with NCS and autotraced about 98% of the structure, with only a couple of gaps/insertions. 3. Running Phaser with the twin option enabled identified the twin fraction easily and resulted in excellent maps for further refinement. If I had been quicker in recognizing the twinning of the data, I probably could have detwinned the data from the start, and Phaser, perhaps in combination with Parrot Buccaneer with NCS, would have arrived at a solution quicker. If you have not used Parrot with NCS pipelined with Buccaneer, give it a try. I was very pleasantly surprised (and impressed) at how efficient this was at getting me to a place where I could start refining the final structure. Your data quality may hamper your ability to solve the structure, especially if you have a large number of overlaps in your high-resolution data. In that case, I wonder if you might fare better by using data cut off to a lower resolution, where the overlap issue is not as severe. With your unit cell, you are probably looking at a long camera distance and thin-slicing to get usable data. That's what we have to do with a 50 x 150 x 250 A unit cell (C2) which is favored by one of our favorite proteins and its variants. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 10/26/2012 8:27 AM, Seijo, Jose A. Cuesta wrote: Hi all, I am dealing with a molecular replacement problem for a 60KDa protein composed of 2 rigid domains joined by a flexible linker which can move relative to each other. Sequence identity for my best model is 46% evenly spread, so in principle this should be a tractable problem. Then the problems start to pile up: a)The unit cell is 56.7Å, 288.5Å, 69.4Å, 90 93.5, 90. Spacegroup P21. Rmerge 12% to 2.4Å. The data also merges relatively well (Rmerge 16%) in P222 with the same a, c and b axes, now of course in that order. In the P21 case, that corresponds to 4 monomers in the asymmetric unit with a solvent content of approx. 50%, giving me 8 domains to find if I separate them. b)The 288 axis means that my data show some overlap in almost all orientations (might be corrected in the future with new datasets), so that my low resolution data are likely unreliable. c)Intensity distributions suggest twinning in either point groups. Actually, they are beyond the perfect twinning case, which I attribute to the reflection overlaps making the strong reflections weaker (integration box too small) and the small stronger (from tails of adjacent strong ones). Of course the latest would mean that the twin fraction estimation is unreliable, but all moments, etc show perfect twin statistics, so I am assuming that there is indeed perfect twinning of some sort. So, the question is, what is the best strategy to deal with this many (4 or 8) body / noisy / twinned problem? I am trying EPMR with many bodies, but I suspect the twinning would throw it out of the right track, and one domain seems to be too little of the diffracting matter to show any sort of discriminations between solutions and non-solutions if do the usual serial searches. I plan to let autotracing programs be the judge of success, but I am not sure of how well those can deal with twinning. Can Arp-Warp use twinned data? Thanks in advance for any tips. Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk
Re: [ccp4bb] Compounded problems for molecular replacement
Hello Jose, Depending on what data integration program you used, trying XDS may help you out a little with spot overlap. Example #3 in my rather out-of-date page: http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacement.html illustrates how you could find 8 domains, especially if you pay attention to the rotation angle values for the candidate domain solutions. This example did not have twinning but did have a little pseudo-centering. This is a 15 year-old example from back when I was using AMORE, so I should clearly rewrite that page. Additionally, if the inter-domain flexibility is restricted to rotation about a single axis, it would be a good idea to rotate your model so that this rotation axis is parallel to the Z axis. This was a method that was exploited with Fab structures (whose elbow angle is a fairly restricted rotation). If so oriented, rotation function peaks relating different domains in the same molecule should show very similar alpha, beta and differ in gamma. Good luck, Phil Jeffrey Princeton On 10/26/12 8:27 AM, Seijo, Jose A. Cuesta wrote: Hi all, I am dealing with a molecular replacement problem for a 60KDa protein composed of 2 rigid domains joined by a flexible linker which can move relative to each other. Sequence identity for my best model is 46% evenly spread, so in principle this should be a tractable problem. Then the problems start to pile up: a)The unit cell is 56.7Å, 288.5Å, 69.4Å, 90 93.5, 90. Spacegroup P21. Rmerge 12% to 2.4Å. The data also merges relatively well (Rmerge 16%) in P222 with the same a, c and b axes, now of course in that order. In the P21 case, that corresponds to 4 monomers in the asymmetric unit with a solvent content of approx. 50%, giving me 8 domains to find if I separate them. b)The 288 axis means that my data show some overlap in almost all orientations (might be corrected in the future with new datasets), so that my low resolution data are likely unreliable. c)Intensity distributions suggest twinning in either point groups. Actually, they are beyond the perfect twinning case, which I attribute to the reflection overlaps making the strong reflections weaker (integration box too small) and the small stronger (from tails of adjacent strong ones). Of course the latest would mean that the twin fraction estimation is unreliable, but all moments, etc show perfect twin statistics, so I am assuming that there is indeed perfect twinning of some sort. So, the question is, what is the best strategy to deal with this many (4 or 8) body / noisy / twinned problem? I am trying EPMR with many bodies, but I suspect the twinning would throw it out of the right track, and one domain seems to be too little of the diffracting matter to show any sort of discriminations between solutions and non-solutions if do the usual serial searches. I plan to let autotracing programs be the judge of success, but I am not sure of how well those can deal with twinning. Can Arp-Warp use twinned data? Thanks in advance for any tips. Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk
