Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
The problem was resolved by Isabel Uson and Giovanna Petrillo using ARCIMBOLDO! The real spacegroup is P21, twinned to look like C2221. I would probably take years to solve that on my own, they did it very fast. This was a difficult problem, and still is, since if I try MR using the correct solution and space group I cannot resolve it. I definitely recommend everybody with difficulties to give Arcimboldo a try. Thank you Isabel! Thank you Sergei Strelkov, your message was very informative as usual. ;) Thank you George M. Sheldrick for the information on how to discrimitate between correct and wrong MR solutions using SHELXE. Best regards, Napo On 11/4/2011 7:42 AM, Sergei Strelkov wrote: Dear Napoleão, Thank you for updating everyone on your efforts, and also acknowledging the advice. I wanted to respond to your question regarding maps. I know that many people who try to figure out whether or not their MR solution is the right one would ask the same question. So first of all if you wonder why you actually get very decently looking maps the answer is a classical one: because 'the phases are more important than amplitudes'. The appearance of your map is defined by your model phases, and hence a good match between the model and the map /may not/ be taken as a sign of a correct solution. Once again: /never ever!/ On the contrary, at least in your coil1.jpg image I clearly see that the density exactly follows the model which is almost entirely poly-Ala. Unless your protein is really poly-Ala this should be alarming. If you had a correct solution they you would hope to see the (difference)density for at least some missing side chains. And a second point. Unless your model contains the complete chain (which is rarely the case, especially for the coiled coils, as discussed already) a sign of the correct solution would be the appearance of extra density near the N- and/or C-terminus of the model. If it is not there, it is almost certainly not a solution. And you should not be worried about the R-factors being very high at this stage. If the solution is correct then you should see at least some extra features in the map. Kind regards, Sergei Thank you all for the replies. Sorry for taking so long to reply, I was actually trying some of your interesting ideas (and I'm still trying). I tried using the low resolution data sets for the molecular replacement (thanks to Yuriy Patskovsky), I also improved and increased my coiled coil database and employed it in many approaches using EPRM (interesting program I was not aware of), which I found to produce lots of data, hopefully addressing at some extent the helixes bent (thanks to Bernhard Rupp). I also tried some more tweaking in Phaser, although not sure if did it properly (thanks to Randy Read). There is no twinning as far as I can tell (thanks to Ed Pozharski for the tip). Using a data set with enough completeness (360 degrees @ Brookhaven) and processing in P1 did not help me because in this space group there is most likely 2-3 helixes in the asymmetric unit, which complicates the problem (and it takes a lot of time for Phaser to run). Automated approaches also did not yield a better result (as far as I can tell). I'm convinced that the space group is C2221, but I may be wrong. Thanks to Sergei Strelkov for the numerous useful suggestions on how to approach the problem. One of the big issues for me is to discriminate between a lot of similarly good density maps. For example: http://www.fullonline.org/coils/coil1.jpg http://www.fullonline.org/coils/coil2.jpg I have hundreds of solutions like these and I think they are all wrong. I couldn't manage to run Arcimboldo, could not find a tutorial on it either. It was highly recommended here (and elsewhere), so I'm definitely willing to give it a try (thanks Isabel Uson). You guys opened my eyes about a series of issues that I should learn about and approach, I'm most thankful for that. Best regards, Napo -- Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven ON2, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Work phone: +32 16 330845 Fax: +32 16 323469 OR +32 16 323460 Mobile: +32 486 294132 Lab pages:http://pharm.kuleuven.be/anafar
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Dear Napoleão, Thank you for updating everyone on your efforts, and also acknowledging the advice. I wanted to respond to your question regarding maps. I know that many people who try to figure out whether or not their MR solution is the right one would ask the same question. So first of all if you wonder why you actually get very decently looking maps the answer is a classical one: because 'the phases are more important than amplitudes'. The appearance of your map is defined by your model phases, and hence a good match between the model and the map /may not/ be taken as a sign of a correct solution. Once again: /never ever!/ On the contrary, at least in your coil1.jpg image I clearly see that the density exactly follows the model which is almost entirely poly-Ala. Unless your protein is really poly-Ala this should be alarming. If you had a correct solution they you would hope to see the (difference)density for at least some missing side chains. And a second point. Unless your model contains the complete chain (which is rarely the case, especially for the coiled coils, as discussed already) a sign of the correct solution would be the appearance of extra density near the N- and/or C-terminus of the model. If it is not there, it is almost certainly not a solution. And you should not be worried about the R-factors being very high at this stage. If the solution is correct then you should see at least some extra features in the map. Kind regards, Sergei Thank you all for the replies. Sorry for taking so long to reply, I was actually trying some of your interesting ideas (and I'm still trying). I tried using the low resolution data sets for the molecular replacement (thanks to Yuriy Patskovsky), I also improved and increased my coiled coil database and employed it in many approaches using EPRM (interesting program I was not aware of), which I found to produce lots of data, hopefully addressing at some extent the helixes bent (thanks to Bernhard Rupp). I also tried some more tweaking in Phaser, although not sure if did it properly (thanks to Randy Read). There is no twinning as far as I can tell (thanks to Ed Pozharski for the tip). Using a data set with enough completeness (360 degrees @ Brookhaven) and processing in P1 did not help me because in this space group there is most likely 2-3 helixes in the asymmetric unit, which complicates the problem (and it takes a lot of time for Phaser to run). Automated approaches also did not yield a better result (as far as I can tell). I'm convinced that the space group is C2221, but I may be wrong. Thanks to Sergei Strelkov for the numerous useful suggestions on how to approach the problem. One of the big issues for me is to discriminate between a lot of similarly good density maps. For example: http://www.fullonline.org/coils/coil1.jpg http://www.fullonline.org/coils/coil2.jpg I have hundreds of solutions like these and I think they are all wrong. I couldn't manage to run Arcimboldo, could not find a tutorial on it either. It was highly recommended here (and elsewhere), so I'm definitely willing to give it a try (thanks Isabel Uson). You guys opened my eyes about a series of issues that I should learn about and approach, I'm most thankful for that. Best regards, Napo -- Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven ON2, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Work phone: +32 16 330845 Fax: +32 16 323469 OR +32 16 323460 Mobile: +32 486 294132 Lab pages: http://pharm.kuleuven.be/anafar
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
We have unintentionally discovered a very simple way of telling whether an MR solution is correct or not, provided that (as in this case) native data have been measured to about 2.1A or better. This uses the current beta-test of SHELXE that does autotracing (available on email request). First rename the PDB file from MR to name.pda and generate a SHELX format file name.hkl, e.g. using Tim Gruene's mtz2hkl, where 'name' may be chosen freely but should be the same for both input files. Then run SHELXE with a large number of autotracing cycles (here 50), e.g. shelxe name.pda -a50 -s0.5 -y2 -s sets the solvent content and -y a resolution limit for generating starting phases. If the .hkl file contains F rather than intensity the -f switch is also required. If the model is wrong the CC value for the trace will gradually decrease as the model disintegrates. If the model is good the CC will increase, and if it reaches 30% or better the structure is solved. In cases with a poor but not entirely wrong starting fragment, the CC may vary erratically for 10-30 cycles before it locks in to the correct solution and the CC increases over three or four cycles to the value for a solved structure (25 to 50%). The solution with the best CC is written to name.pdb and its phases to name.phs for input to e.g. Coot. George On Fri, Nov 04, 2011 at 10:42:27AM +0100, Sergei Strelkov wrote: Dear Napoleão, Thank you for updating everyone on your efforts, and also acknowledging the advice. I wanted to respond to your question regarding maps. I know that many people who try to figure out whether or not their MR solution is the right one would ask the same question. So first of all if you wonder why you actually get very decently looking maps the answer is a classical one: because 'the phases are more important than amplitudes'. The appearance of your map is defined by your model phases, and hence a good match between the model and the map may not be taken as a sign of a correct solution. Once again: never ever! On the contrary, at least in your coil1.jpg image I clearly see that the density exactly follows the model which is almost entirely poly-Ala. Unless your protein is really poly-Ala this should be alarming. If you had a correct solution they you would hope to see the (difference)density for at least some missing side chains. And a second point. Unless your model contains the complete chain (which is rarely the case, especially for the coiled coils, as discussed already) a sign of the correct solution would be the appearance of extra density near the N- and/or C-terminus of the model. If it is not there, it is almost certainly not a solution. And you should not be worried about the R-factors being very high at this stage. If the solution is correct then you should see at least some extra features in the map. Kind regards, Sergei Thank you all for the replies. Sorry for taking so long to reply, I was actually trying some of your interesting ideas (and I'm still trying). I tried using the low resolution data sets for the molecular replacement (thanks to Yuriy Patskovsky), I also improved and increased my coiled coil database and employed it in many approaches using EPRM (interesting program I was not aware of), which I found to produce lots of data, hopefully addressing at some extent the helixes bent (thanks to Bernhard Rupp). I also tried some more tweaking in Phaser, although not sure if did it properly (thanks to Randy Read). There is no twinning as far as I can tell (thanks to Ed Pozharski for the tip). Using a data set with enough completeness (360 degrees @ Brookhaven) and processing in P1 did not help me because in this space group there is most likely 2-3 helixes in the asymmetric unit, which complicates the problem (and it takes a lot of time for Phaser to run). Automated approaches also did not yield a better result (as far as I can tell). I'm convinced that the space group is C2221, but I may be wrong. Thanks to Sergei Strelkov for the numerous useful suggestions on how to approach the problem. One of the big issues for me is to discriminate between a lot of similarly good density maps. For example: http://www.fullonline.org/coils/coil1.jpg http://www.fullonline.org/coils/coil2.jpg I have hundreds of solutions like these and I think they are all wrong. I couldn't manage to run Arcimboldo, could not find a tutorial on it either. It was highly recommended here (and elsewhere), so I'm definitely willing to give it a try (thanks Isabel Uson). You guys opened my eyes about a series of issues that I should learn about and approach, I'm most thankful for that. Best regards, Napo -- Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Thank you all for the replies. Sorry for taking so long to reply, I was actually trying some of your interesting ideas (and I'm still trying). I tried using the low resolution data sets for the molecular replacement (thanks to Yuriy Patskovsky), I also improved and increased my coiled coil database and employed it in many approaches using EPRM (interesting program I was not aware of), which I found to produce lots of data, hopefully addressing at some extent the helixes bent (thanks to Bernhard Rupp). I also tried some more tweaking in Phaser, although not sure if did it properly (thanks to Randy Read). There is no twinning as far as I can tell (thanks to Ed Pozharski for the tip). Using a data set with enough completeness (360 degrees @ Brookhaven) and processing in P1 did not help me because in this space group there is most likely 2-3 helixes in the asymmetric unit, which complicates the problem (and it takes a lot of time for Phaser to run). Automated approaches also did not yield a better result (as far as I can tell). I'm convinced that the space group is C2221, but I may be wrong. Thanks to Sergei Strelkov for the numerous useful suggestions on how to approach the problem. One of the big issues for me is to discriminate between a lot of similarly good density maps. For example: http://www.fullonline.org/coils/coil1.jpg http://www.fullonline.org/coils/coil2.jpg I have hundreds of solutions like these and I think they are all wrong. I couldn't manage to run Arcimboldo, could not find a tutorial on it either. It was highly recommended here (and elsewhere), so I'm definitely willing to give it a try (thanks Isabel Uson). You guys opened my eyes about a series of issues that I should learn about and approach, I'm most thankful for that. Best regards, Napo
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Hi Napo, i am sorry if you already answered this, why are so sure your solutions are wrong? Your maps do not look that bad. What kind of R's do you get? Are you not happy with the packing of your coils?
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Dear Napoleão, I will try to summarize our experience and give some suggestions. Few reasons why MR with coiled coils can be very tricky, such as their asymmetric shape and their ability to overlap onto themselves upon a shift and rotation (for a heptad-based coiled coil, this would be a shift by 7 residues) have been already mentioned. And I can add two more. First, we very often see that coiled coils get bent due to crystal contacts. This means that the conformation may be quite different compared to your search model. Second, many coiled coil sequences were seen to assemble into structures different to what they were supposed to be. Examples I know include a trimer when a dimer was expected, an antiparallel coiled coil when a parallel one was expected, a monomer when a a dimer was expected, chains offset with respect to each other, etc etc We were able to phase several short (40-60 residues) coiled coils by MR in the past. The paper Strelkov SV et al (2002) EMBO Journal describes two such cases (please look at the Methods section which provides a fairly detailed explanation of what we did). Both were done with Molrep. I do have to say that we also failed on MR miserably in many other cases, whatever we tried. One recent example of a coiled coil fragment that assembled to something entirely different than we expected it to, is in Nicolet S et al (2010) J. Struct. Biol. There, we really had to use heavy atoms. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. You have collected many data sets and you are still not sure about the space group - ? Have you looked at the systematic absences carefully? If you are still missing the (00l) axis then you probably could try collecting it again while considering the orientation of your crystal. Knowing the correct space group is a valuable information. Yes there are cases when you can not distinguish between two space groups from the diffraction pattern (e.g. P61 and P65) and then you /have/ to run your MR search twice in both groups. Yes modern programs will do it almost by default for you. But if you do know your correct space group (for instance C2221 and not C222) then when you do the MR search in the two space groups you /may/ (with some luck) see better results with the first than with the second. This /may/ be a hint that your C2221 solution is correct. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. As mentioned already, is really advisable to try other MR programs (MolRep, epmr etc), as in fact they are all based on different algorithms. I'm using a 80% identity coiled coil helix as search model. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. From your description I am guessing that you use a monomeric helix currently - ? Indeed you really should try different models, anything you can get your hands on. In fact this is a number one factor to get MR to work. On one hand, you should indeed try shortening your model (even systematically trimming one residue at a time). Do not hesitate to use much shorter models (less than half of your full helix). You can trim the wrong side chains but I would not advise chopping off all of them. With a short helix, once you have the first candidate solution, try searching for a second copy with the packing penalty switched off. If you get another helix overlapping with your first solution (with some offset) this may be a sign of success. (see the EMBO J paper) On the other hand, our experience shows that in many cases you can only get a solution if you use a full coiled coil (dimer, trimer,...) and not a monomeric helix. If your real structure is a non-crystallographic dimer etc, then you should definitely search with a dimer etc. If your oligomer is due to a crystallographic axis, then you may try this as well, after switching off the packing penalty. But beware that the oligomer will almost certainly land on the axis, even for a wrong solution. Hope this will help, and best of luck with your MR searches -- which are fun, since they still require some thinking! Sergei -- Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven ON2, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Work phone: +32 16 330845 Fax: +32 16 323469 OR +32 16 323460 Mobile: +32 486 294132 Lab pages: http://pharm.kuleuven.be/anafar
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Hello, James Holton can probably tell more about this, but it is possible to create a library of potential coiled coil structures with differences in number of residues, superhelical radius, and residues per superhelical turn. A library of 300 theoretical coiled coils was generated and, in conjunction with EPMR, was used successfully to solve the structure of a KCNQ tetrameric coiled coil. See: Howard et al (2007) Neuron 53(5),663-675. And I second Sergei Strelkov's comment: what should be an expected tetramer could show up as a trimer, etc... so you may want to check via ultracentrifugation that you have the expected stoichiometry. Regards, Filip On Mon, Oct 17, 2011 at 10:09 AM, Napoleão Valadares n...@ifsc.usp.brwrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Dear Napoleão, we solved this type of problem in a paper of 2004 (J. Mol. Biol. (2004) 342, 275–287) with the using of EPMR which use a evolutionary search algorithm, I will send you the paper later. It was 36aa dimeric coiled-coil and we had a lot of molecular replacement problems with other tested molecular replacement programs. http://www.epmr.info/UsersGuide.html Good luck.
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Just to add to what has been said (written) before: coiled coiled or simply helices can be very problematic for M.R.. Human sacrifice has never given any positive result (as reported in the literature as far as I know), but heavy atom sacrifice could be attempted (heavy atom includes in my view atoms that are used for SAD, MAD - such as S, Se... - in addition to Au, Pt...) in parallel to your M.R. searches which may never provide you with an acceptable solution. Fred. Napoleão Valadares wrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo
[ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
If for example your presumed coil is differently bent than your search probe, overall rmsd is high and MR will likely fail. But maybe ARCIMBOLDO with helix fragments might do at 1.65A? George or Isabel will have a better answer. Good luck, BR PS: And may San Matheus de las Coefficientes listen to your prayers... :-) -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Napoleão Valadares Sent: Monday, October 17, 2011 10:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Did you check for twinning? The most radical approach is to reprocess in P1 and see what R-values you get On Mon, 2011-10-17 at 15:09 -0200, Napoleão Valadares wrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
On Mon, Oct 17, 2011 at 10:09 AM, Napoleão Valadares n...@ifsc.usp.brwrote: I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. Did you try running an automated building program (phenix.autobuild or ARP/wARP)? If the solutions are real, the software should do a very good job, much better than refinement alone. Otherwise, I agree ARCIMBOLDO sounds like a good choice here. -Nat
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. This strongly suggests a space group issue. If the systematic absences are compelling, you should try to drop it down to P2 first. Look at your packing in the C222(1). If you change the direction of one of the coils relative to the other, will it break the orthorhombic symmetry and drop it to monoclinic? This may provide a clue to the space group you should try. James On Oct 17, 2011, at 11:09 AM, Napoleão Valadares wrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
I should have said not compelling. James On Oct 17, 2011, at 12:40 PM, James Stroud wrote: The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. This strongly suggests a space group issue. If the systematic absences are compelling, you should try to drop it down to P2 first. Look at your packing in the C222(1). If you change the direction of one of the coils relative to the other, will it break the orthorhombic symmetry and drop it to monoclinic? This may provide a clue to the space group you should try.
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Hi, I agree that Arcimboldo is a good idea, especially at such good resolution (assuming your space group is correct, an issue that others have commented on). But you could also try, directly, something that Isabel Uson has pointed out about molecular replacement with helices in Phaser. For such asymmetric objects, the rotational sampling tends to be too coarse to sample the orientations rotated around an axis perpendicular to the helix axis. So you could look at the rotational sampling chosen by Phaser and override it with something, say, 1/2 to 2/3 as large. Another issue is that, with a long helix, there will be many nearly-correct solutions offset by partial turns. So the sequence register could be wrong. Good luck! Randy Read On 17 Oct 2011, at 18:09, Napoleão Valadares wrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 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
