Re: [ccp4bb] Disorder or poor phases?
Nothing profound to add to this interesting discussion, but I too would like to plug FobsA - FobsB type maps - when A and B are similar but not quite the same.. It is prudent to omit the interesting parts of model A (or B) - whichever you use to calculate the PHIC and FOM - but the peaks and pits often clear up ambiguity brilliantly. You need to CAD the two data sets together, and make sure both Fobs are on the same scale.. Eleanor On 13 April 2012 19:50, Gloria Borgstahl gborgst...@gmail.com wrote: a recent experience in our lab with molecular replacement (wt and disordered point mutant; same space group and unit cell) was solved with a combination of two methods. 1. We made omit maps in the disordered region at several lower resolutions. The region became interpretable after suffereing through these maps, building residue by residue and refinement. 2. Then we had the bright idea to make Fwt-Fmutant maps to confirm our interpretation. Happily this map did confirm the unexpected large structural changed caused by a point mutant. On Fri, Apr 13, 2012 at 1:31 PM, James Holton jmhol...@lbl.gov wrote: Francis, I think in the cases you describe the region in question is disordered. Time and time again I have users coming to my beamline wanting to clean up a questionable region by getting experimental phases. Ahh! If only I had a nickle for each one. Oh wait, I suppose I kind of do? I take that back! Go MAD everyone! Much as I hate to discourage people from using my favorite technique, Tim is right: phases are not region-specific in electron density maps. Dale does make a good point that there is such a thing as model bias and one can argue that experimental phases don't have it. But, this is only true if you have not yet applied solvent flattening. How long has it been since you looked at a raw experimentally-phased map (before solvent flattening)? I'm willing to bet a while. With very few exceptions, raw experimental phases are lousy. We have actually become quite dependent on density modification to clean them up. In fact, solvent flattening is the only reason why SAD works at all. However, you CAN use anomalous differences to clear up disordered regions in a different way. Something I started calling SeMet scanning a number of years ago. A few of my users have done this, and a good example of it is Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate residues in the disordered region one at a time to SeMet, and look at phased anomalous difference Fourier (PADF) maps. These maps are surprisingly clear, even when the anomalous difference signal is so weak as to make experimental phasing hopeless. Yes, the best phases to use for PADF maps are model phases, but, as always, it is prudent to refine the model after omitting the thing you are looking for before calculating such phases. Another way to get residue-specific labeling for low-resolution chain tracing is radiation damage. If you expose for the right amount of time, Asp and Glu side chains will be specifically burnt off, but not Asn and Gln. You will also see Met loosing its head, etc. So, as long as you have read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to guide sequence assignment, even at 4.5 A and worse. Anyway, when it comes to the question of is it disordered or is it model bias?, I think it is usually the former. It is very difficult to make model bias suppress a region that is actually well-ordered. Try it! After all, this is the whole reason why we bother looking at fo-fc maps. Then again, it is always possible to have a model so bad that the phase error is enough to squash anything. An excellent example of this can be found in the Book of Fourier. Taking amplitudes from the image of a cat, you can see what happens when you use the phases of a duck: http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif as opposed to what happens if you use the phases of a manx: http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif A manx is a species of cat that doesn't have a tail, so no animals were harmed in obtaining these phases. My point here is that the cat's tail can be seen quite readily in the 2fo-fc map if most of the structure is already right, but if your model is completely unrelated to the true structure (fitting a duck into a cat-shaped hole), then everything is in the noise. Real structures are usually somewhere between these two extremes, and I think an important shortcoming in modern crystallography is that we don't have a good quantitative description of this middle-ground. We all like to think we know what model bias is, but we don't exactly have units for it. Should we be using a scale of 0 to 1? Or perhaps duck to cat? Yes, I know we have figure of merit, but FOM is not region-specific. In
Re: [ccp4bb] Disorder or poor phases?
Francis, I think in the cases you describe the region in question is disordered. Time and time again I have users coming to my beamline wanting to clean up a questionable region by getting experimental phases. Ahh! If only I had a nickle for each one. Oh wait, I suppose I kind of do? I take that back! Go MAD everyone! Much as I hate to discourage people from using my favorite technique, Tim is right: phases are not region-specific in electron density maps. Dale does make a good point that there is such a thing as model bias and one can argue that experimental phases don't have it. But, this is only true if you have not yet applied solvent flattening. How long has it been since you looked at a raw experimentally-phased map (before solvent flattening)? I'm willing to bet a while. With very few exceptions, raw experimental phases are lousy. We have actually become quite dependent on density modification to clean them up. In fact, solvent flattening is the only reason why SAD works at all. However, you CAN use anomalous differences to clear up disordered regions in a different way. Something I started calling SeMet scanning a number of years ago. A few of my users have done this, and a good example of it is Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate residues in the disordered region one at a time to SeMet, and look at phased anomalous difference Fourier (PADF) maps. These maps are surprisingly clear, even when the anomalous difference signal is so weak as to make experimental phasing hopeless. Yes, the best phases to use for PADF maps are model phases, but, as always, it is prudent to refine the model after omitting the thing you are looking for before calculating such phases. Another way to get residue-specific labeling for low-resolution chain tracing is radiation damage. If you expose for the right amount of time, Asp and Glu side chains will be specifically burnt off, but not Asn and Gln. You will also see Met loosing its head, etc. So, as long as you have read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to guide sequence assignment, even at 4.5 A and worse. Anyway, when it comes to the question of is it disordered or is it model bias?, I think it is usually the former. It is very difficult to make model bias suppress a region that is actually well-ordered. Try it! After all, this is the whole reason why we bother looking at fo-fc maps. Then again, it is always possible to have a model so bad that the phase error is enough to squash anything. An excellent example of this can be found in the Book of Fourier. Taking amplitudes from the image of a cat, you can see what happens when you use the phases of a duck: http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif as opposed to what happens if you use the phases of a manx: http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif A manx is a species of cat that doesn't have a tail, so no animals were harmed in obtaining these phases. My point here is that the cat's tail can be seen quite readily in the 2fo-fc map if most of the structure is already right, but if your model is completely unrelated to the true structure (fitting a duck into a cat-shaped hole), then everything is in the noise. Real structures are usually somewhere between these two extremes, and I think an important shortcoming in modern crystallography is that we don't have a good quantitative description of this middle-ground. We all like to think we know what model bias is, but we don't exactly have units for it. Should we be using a scale of 0 to 1? Or perhaps duck to cat? Yes, I know we have figure of merit, but FOM is not region-specific. In my experience, as long as you have ~50% of the electrons in the right place (and none of them in the wrong place), then you can generally trust that the biggest difference feature in the fo-fc map is real, and build from there. As the model becomes more complete, the phases should continue to get better, not worse. Eventually, this does break down, although I'm not really sure why. With small molecules, the maximum fo-fc peak keeps getting bigger (on a sigma scale) as you add more and more atoms, and the biggest one you will ever see is the last one. For macromolecules, the difference features keep getting smaller and smaller as you build. Perhaps small errors (like non-Gaussian atomic displacement distributions being modeled as Gaussians) slowly accumulate? Perhaps there are other sources of systematic error that we don't yet fully understand? Eventually, for whatever reason, you stop building. Having electrons in the wrong place is about twice as bad as not having them at all, which I think is why we trim models so aggressively for molecular replacement, and also why we are so reticent to model in things that we are not sure about. Disordered regions, of course, will
Re: [ccp4bb] Disorder or poor phases?
a recent experience in our lab with molecular replacement (wt and disordered point mutant; same space group and unit cell) was solved with a combination of two methods. 1. We made omit maps in the disordered region at several lower resolutions. The region became interpretable after suffereing through these maps, building residue by residue and refinement. 2. Then we had the bright idea to make Fwt-Fmutant maps to confirm our interpretation. Happily this map did confirm the unexpected large structural changed caused by a point mutant. On Fri, Apr 13, 2012 at 1:31 PM, James Holton jmhol...@lbl.gov wrote: Francis, I think in the cases you describe the region in question is disordered. Time and time again I have users coming to my beamline wanting to clean up a questionable region by getting experimental phases. Ahh! If only I had a nickle for each one. Oh wait, I suppose I kind of do? I take that back! Go MAD everyone! Much as I hate to discourage people from using my favorite technique, Tim is right: phases are not region-specific in electron density maps. Dale does make a good point that there is such a thing as model bias and one can argue that experimental phases don't have it. But, this is only true if you have not yet applied solvent flattening. How long has it been since you looked at a raw experimentally-phased map (before solvent flattening)? I'm willing to bet a while. With very few exceptions, raw experimental phases are lousy. We have actually become quite dependent on density modification to clean them up. In fact, solvent flattening is the only reason why SAD works at all. However, you CAN use anomalous differences to clear up disordered regions in a different way. Something I started calling SeMet scanning a number of years ago. A few of my users have done this, and a good example of it is Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate residues in the disordered region one at a time to SeMet, and look at phased anomalous difference Fourier (PADF) maps. These maps are surprisingly clear, even when the anomalous difference signal is so weak as to make experimental phasing hopeless. Yes, the best phases to use for PADF maps are model phases, but, as always, it is prudent to refine the model after omitting the thing you are looking for before calculating such phases. Another way to get residue-specific labeling for low-resolution chain tracing is radiation damage. If you expose for the right amount of time, Asp and Glu side chains will be specifically burnt off, but not Asn and Gln. You will also see Met loosing its head, etc. So, as long as you have read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to guide sequence assignment, even at 4.5 A and worse. Anyway, when it comes to the question of is it disordered or is it model bias?, I think it is usually the former. It is very difficult to make model bias suppress a region that is actually well-ordered. Try it! After all, this is the whole reason why we bother looking at fo-fc maps. Then again, it is always possible to have a model so bad that the phase error is enough to squash anything. An excellent example of this can be found in the Book of Fourier. Taking amplitudes from the image of a cat, you can see what happens when you use the phases of a duck: http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif as opposed to what happens if you use the phases of a manx: http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif A manx is a species of cat that doesn't have a tail, so no animals were harmed in obtaining these phases. My point here is that the cat's tail can be seen quite readily in the 2fo-fc map if most of the structure is already right, but if your model is completely unrelated to the true structure (fitting a duck into a cat-shaped hole), then everything is in the noise. Real structures are usually somewhere between these two extremes, and I think an important shortcoming in modern crystallography is that we don't have a good quantitative description of this middle-ground. We all like to think we know what model bias is, but we don't exactly have units for it. Should we be using a scale of 0 to 1? Or perhaps duck to cat? Yes, I know we have figure of merit, but FOM is not region-specific. In my experience, as long as you have ~50% of the electrons in the right place (and none of them in the wrong place), then you can generally trust that the biggest difference feature in the fo-fc map is real, and build from there. As the model becomes more complete, the phases should continue to get better, not worse. Eventually, this does break down, although I'm not really sure why. With small molecules, the maximum fo-fc peak keeps getting bigger (on a sigma scale) as you add more and more atoms, and the biggest one you will ever see is the last one. For macromolecules, the
Re: [ccp4bb] Disorder or poor phases?
On 4/10/2012 10:44 PM, Kay Diederichs wrote: Hi Dale, my experience is that high-B regions may become visible in maps only late in refinement. So my answer to the original poster would be - both global reciprocal-space (phase quality) and local real-space (high mobility) features contribute to a region not appearing ordered in the map. This would be supported by your experience if those residues that you could not model in 3BCL had high (or at least higher) B-factors compared to the rest of the model. Is that so? Actually the residues I couldn't model in 3BCL had no B's... :) Seriously, the residues that appeared for 4BCL did have B values much higher than average. Their density was weak in the best of circumstances and more susceptible to obliteration by the distortions caused by imprecision in the phases. I don't really want to describe this as phase error as that phrase conjures notions of large changes in phase. The R value only dropped from 18.9% to 17.8% from 3BCL to 4BCL. I don't expect there were huge differences in the phase angles, but the differences were enough. Dale best, Kay
Re: [ccp4bb] Disorder or poor phases?
Hello, Kay is absolutely right. Just to make this clear: We all know that in many cases, you start out with poor phases (i.e. a weak SIR/MIR/MAD or a borderline replacement model) and your density is modest. The prudent thing to do at this stage is, to build only things you trust and have a look at the improved density. Well, we all know also, that an improved density means in most cases a density with improved phases. The term disorder means, a region of higher uncertainty. Logically, the more information you have (more actual data points - i.e. reflections == resolution/completeness; more reliable Fs; etc.; _better phases_) the better you can pinpoint these areas. The phase is a magnitude we cannot measure, but that affects the density the most. We determine it through refinement (which encompasses density interpretation and computational optimization of atomic parameters with regards to the reflection data). Gedankenexperiment: If you collect data on a crystal, let's say on a sealed tube from 1950 with a photon counter, and you collect the same data from the same crystal on a modern synchrotron with a PAD, you might find certain areas of your molecule disordered that you might be able to interpret with (more) data collected from the better collected data. Probably more so - if you have the same amount of data and poorer or better phases, you have a similar problem. My point being: the term disorder is related to the amount of data you have (be it collected (I's) or deduced (phi's)). With very few exceptions (see for example the paper for 1M1N), it's not the method (diffraction) that tricks us, it's just the amount of information that we have, that prevents us from building complete models. Most importantly, the term disordered - as used in macromolecular terminology - depends on resolution /and/ quality of the phases. (As a side note: What we call alternative conformations in macromolecular crystallography is called disorder in small molecule crystallography. I don't know what the SM word for the MM disorder is...) Cheers, Jens On Wed, 2012-04-11 at 06:44 +0100, Kay Diederichs wrote: Hi Dale, my experience is that high-B regions may become visible in maps only late in refinement. So my answer to the original poster would be - both global reciprocal-space (phase quality) and local real-space (high mobility) features contribute to a region not appearing ordered in the map. This would be supported by your experience if those residues that you could not model in 3BCL had high (or at least higher) B-factors compared to the rest of the model. Is that so? best, Kay
Re: [ccp4bb] Disorder or poor phases?
I am not sure anyone looks at plain 2fo-fc maps anymore - it almost always (at least since the beginning of the 3rd millennium) implies 2mfo-Dfc ML maps. Detailed explanation of coefficients and their relation to ML sigma A are in R.Read papers, BMC, Bricogne, etc pp BR, On Tue, Apr 10, 2012 at 8:38 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Francis, the phases calculated from the model affect the whole unit cell hence it is more likely this is real(-space, local) disorder rather than poor phases. Regards, Tim P.S.: The author should not look at an 2fofc-map but a sigma-A-weighted map to reduce model bias. On 04/10/12 17:22, Francis E Reyes wrote: Hi all, Assume that the diffraction resolution is low (say 3.0A or worse) and the model (a high resolution homologue, from 2A xray data is available) was docked into experimental phases (say 4A or worse) and extended to the 3.0A data using refinement (the high resolution model as a source of restraints). There are some conformational differences between the high resolution model and the target crystal. The author observes that in the 2fofc map at 3A, most of the model shows reasonable density, but for a stretch of backbone the density is weak. Is the weakness of the density in this region because of disorder or bad model phases? Would love people's thoughts on this one, F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPhFP3UxlJ7aRr7hoRAj4HAKDpHCsN+tBKhDAcOYmIe5c58ThG+gCeMujG pAJxRNuJHE4+oFRPSYx4bnc= =s3uw -END PGP SIGNATURE- -- - Bernhard Rupp (Hofkristallrat a. D) 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - The hard part about playing chicken is to know when to flinch -
[ccp4bb] Disorder or poor phases?
Hi all, Assume that the diffraction resolution is low (say 3.0A or worse) and the model (a high resolution homologue, from 2A xray data is available) was docked into experimental phases (say 4A or worse) and extended to the 3.0A data using refinement (the high resolution model as a source of restraints). There are some conformational differences between the high resolution model and the target crystal. The author observes that in the 2fofc map at 3A, most of the model shows reasonable density, but for a stretch of backbone the density is weak. Is the weakness of the density in this region because of disorder or bad model phases? Would love people's thoughts on this one, F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Disorder or poor phases?
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Francis, the phases calculated from the model affect the whole unit cell hence it is more likely this is real(-space, local) disorder rather than poor phases. Regards, Tim P.S.: The author should not look at an 2fofc-map but a sigma-A-weighted map to reduce model bias. On 04/10/12 17:22, Francis E Reyes wrote: Hi all, Assume that the diffraction resolution is low (say 3.0A or worse) and the model (a high resolution homologue, from 2A xray data is available) was docked into experimental phases (say 4A or worse) and extended to the 3.0A data using refinement (the high resolution model as a source of restraints). There are some conformational differences between the high resolution model and the target crystal. The author observes that in the 2fofc map at 3A, most of the model shows reasonable density, but for a stretch of backbone the density is weak. Is the weakness of the density in this region because of disorder or bad model phases? Would love people's thoughts on this one, F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPhFP3UxlJ7aRr7hoRAj4HAKDpHCsN+tBKhDAcOYmIe5c58ThG+gCeMujG pAJxRNuJHE4+oFRPSYx4bnc= =s3uw -END PGP SIGNATURE-
Re: [ccp4bb] Disorder or poor phases?
Dale Thank you for the case study. I will certainly remember it when I next see: I don't see density for these atoms therefore they must be disordered. You do mention though, that when you were able to assign the sequence to the beta sheets, that the loop regions became clear. I consider the case (which a majority of cases seem to be), where the author has built and sequence assigned 95% of the ASU, but is unable to model a loop region. One possibility is that the loop is truly disordered (95% of the ASU is built and is presumably right), the other possibility is that there's an inherent error in the existing structure that is affecting the interpretation of the loop region. The errors are probably extremely subtle and distributed throughout the model (think of the improvements DEN refinement gave for the rerefinement of p97). I guess in either case, because of the dependency of the map on the existing set of phases it's difficult to determine whether it's truly disordered or not. P.S.: The author should not look at an 2fofc-map but a sigma-A-weighted map to reduce model bias. Tim, I assume a sigmaA weighted 2Fo-Fc map (which I believe is the default for most crystallographic refinement packages). F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Disorder or poor phases?
Dear Dale, There is perhaps a third factor in the progress you were able to make, namely the improvement in the refinement programs. Your first results were probably obtained with a least-squares-based program, while the more recent would have come from maximum-likelihood-based ones. The difference lies in the quality of the phase information produced from the model through comparison of Fo and Fc, with much greater bias-correction capabilities in the ML approach. Here, it removed the bias towards some regions being absent in the model, and made them no longer be absent in the maps. So it is a question of the quality of the phase information. With best wishes, Gerard. -- On Tue, Apr 10, 2012 at 12:00:28PM -0700, Dale Tronrud wrote: The phases do have effects all over the unit cell but that does not prevent them from constructively and destructively interfering with one another in particular locations. Some years ago I refined a model of the bacteriochlorophyll containing protein to a 1.9 A data set when the sequence of that protein was unknown. This is primarily a beta sheet protein and a number of the loops between the strands were disordered. Later the amino acid sequence was determined and I finished the refinement after building in these corrections. The same data set was used, but a number of the loops had become ordered. While the earlier model (3BCL) had 357 amino acids the final model (4BCL) had 366. These nine amino acids didn't become ordered over the intervening years. They were just as ordered when I was building w/o a sequence, it is just that I couldn't see how to build them based on the map's appearance. One possibility is that the density for these residues was weak and the noise (that was uniform over the entire map) obliterated their signal where it only obscured the stronger density. Another possibility is that the better model had a better match of the low resolution F's and less intense ripples radiating from the surface of the molecule, resulting in things sticking out being less affected. Whatever the details, the density for these amino acids were too weak to model with the poorer model phases and became buildable with better phases. The fact that they could not be seen in the early map was not an indication that they were disordered. The first six amino acids of this protein have never been seen in any map, including the 1.3 A resolution model 3EOJ (which by all rights should have been called 5BCL ;-) ). These residues appear to be truly disordered. Going back to 3BCL - The map for this model is missing density for a number of residues of which we know some are disordered and some simply unmodelable because of the low quality of the phases. I don't know of a way, looking at that map alone, of deciding which is which. Because of this observation I don't believe it is supportable to say I don't see density for these atoms therefore they must be disordered. Additional evidence is required. Dale Tronrud On 04/10/12 08:38, Tim Gruene wrote: Dear Francis, the phases calculated from the model affect the whole unit cell hence it is more likely this is real(-space, local) disorder rather than poor phases. Regards, Tim P.S.: The author should not look at an 2fofc-map but a sigma-A-weighted map to reduce model bias. On 04/10/12 17:22, Francis E Reyes wrote: Hi all, Assume that the diffraction resolution is low (say 3.0A or worse) and the model (a high resolution homologue, from 2A xray data is available) was docked into experimental phases (say 4A or worse) and extended to the 3.0A data using refinement (the high resolution model as a source of restraints). There are some conformational differences between the high resolution model and the target crystal. The author observes that in the 2fofc map at 3A, most of the model shows reasonable density, but for a stretch of backbone the density is weak. Is the weakness of the density in this region because of disorder or bad model phases? Would love people's thoughts on this one, F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Disorder or poor phases?
Dear Gerard, No, the updated model (4BCL) was published in 1993 (although apparently not deposited until 1998 - What was wrong with me?) Both were refined with that classic least-squares program TNT. I hope there was some improvement in the software between 1986 and 1993, and I always tried to work with the most recent version, but there wasn't a switch in target function. I agree that the distortions in these maps would have been less if an ML approach had been used and perhaps the location of the disordered residues would have been apparent earlier in the process. Maybe this sort of problem will not be seen again at 1.9 A resolution. My goal was simply to provide an example where errors due to model phases didn't distribute evenly throughout the map but had greater consequence in some locations. Dale On 04/10/12 13:45, Gerard Bricogne wrote: Dear Dale, There is perhaps a third factor in the progress you were able to make, namely the improvement in the refinement programs. Your first results were probably obtained with a least-squares-based program, while the more recent would have come from maximum-likelihood-based ones. The difference lies in the quality of the phase information produced from the model through comparison of Fo and Fc, with much greater bias-correction capabilities in the ML approach. Here, it removed the bias towards some regions being absent in the model, and made them no longer be absent in the maps. So it is a question of the quality of the phase information. With best wishes, Gerard. -- On Tue, Apr 10, 2012 at 12:00:28PM -0700, Dale Tronrud wrote: The phases do have effects all over the unit cell but that does not prevent them from constructively and destructively interfering with one another in particular locations. Some years ago I refined a model of the bacteriochlorophyll containing protein to a 1.9 A data set when the sequence of that protein was unknown. This is primarily a beta sheet protein and a number of the loops between the strands were disordered. Later the amino acid sequence was determined and I finished the refinement after building in these corrections. The same data set was used, but a number of the loops had become ordered. While the earlier model (3BCL) had 357 amino acids the final model (4BCL) had 366. These nine amino acids didn't become ordered over the intervening years. They were just as ordered when I was building w/o a sequence, it is just that I couldn't see how to build them based on the map's appearance. One possibility is that the density for these residues was weak and the noise (that was uniform over the entire map) obliterated their signal where it only obscured the stronger density. Another possibility is that the better model had a better match of the low resolution F's and less intense ripples radiating from the surface of the molecule, resulting in things sticking out being less affected. Whatever the details, the density for these amino acids were too weak to model with the poorer model phases and became buildable with better phases. The fact that they could not be seen in the early map was not an indication that they were disordered. The first six amino acids of this protein have never been seen in any map, including the 1.3 A resolution model 3EOJ (which by all rights should have been called 5BCL ;-) ). These residues appear to be truly disordered. Going back to 3BCL - The map for this model is missing density for a number of residues of which we know some are disordered and some simply unmodelable because of the low quality of the phases. I don't know of a way, looking at that map alone, of deciding which is which. Because of this observation I don't believe it is supportable to say I don't see density for these atoms therefore they must be disordered. Additional evidence is required. Dale Tronrud On 04/10/12 08:38, Tim Gruene wrote: Dear Francis, the phases calculated from the model affect the whole unit cell hence it is more likely this is real(-space, local) disorder rather than poor phases. Regards, Tim P.S.: The author should not look at an 2fofc-map but a sigma-A-weighted map to reduce model bias. On 04/10/12 17:22, Francis E Reyes wrote: Hi all, Assume that the diffraction resolution is low (say 3.0A or worse) and the model (a high resolution homologue, from 2A xray data is available) was docked into experimental phases (say 4A or worse) and extended to the 3.0A data using refinement (the high resolution model as a source of restraints). There are some conformational differences between the high resolution model and the target crystal. The author observes that in the 2fofc map at 3A, most of the model shows reasonable density, but for a stretch of backbone the density is weak. Is the weakness of the density in this region because of disorder or bad model phases? Would
Re: [ccp4bb] Disorder or poor phases?
Hi Dale, my experience is that high-B regions may become visible in maps only late in refinement. So my answer to the original poster would be - both global reciprocal-space (phase quality) and local real-space (high mobility) features contribute to a region not appearing ordered in the map. This would be supported by your experience if those residues that you could not model in 3BCL had high (or at least higher) B-factors compared to the rest of the model. Is that so? best, Kay
