Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
1) You say 3000 reflections - 400 parameters, but you arent including the restraints as observations - refinement and rebuilding IS possible at 3.2A.. 2) B factors not unreasonable for the data.. You may want to use fixed Bs with TLS or restrain Bs strictly.. 3) How can your FreeR and r factor be so close - that MUST be wrong.. 4) You can tighten the geometry with coot or with stricter restraints. Maybe set the occs of the bad residues to 0.00 and see what they look like after refining the other better determined parts? They could then perhaps be rebuilt with more acceptable geometry. Eleanor If you are sure of On 01/06/2012 08:14 AM, 商元 wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
On Fri, Jan 06, 2012 at 04:23:03PM +0800, ?? wrote: Also, there is one more information I forgot to mention---I also have the NMR assignment(HNCACB spectrum) of the protein, is it possible to combine the NMR data in my refinement? Yes - we did something a few years back for the structure of the human voltage-dependent anion channel (slightly more reflections, but lower resolution) using a combination of Se-MET phases (SHARP), NMR and secondary-structure restraints in refinement (BUSTER). See Monika Bayrhuber, Thomas Meins, Michael Habeck, Stefan Becker, Karin Giller, Saskia Villinger, Clemens Vonrhein, Christian Griesinger, Markus Zweckstetter, Kornelius Zeth(2008): Structure of the human voltage dependent anion channel. Proc. Nat. Acad. Sci. USA 105: 15370-15375. or http://www.pnas.org/content/105/40/15370.full It contains a fair amount of background info about methods. Cheers Clemens Regards, On Fri, Jan 6, 2012 at 4:14 PM, ?? shangyuan5...@gmail.com wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Also, there is one more information I forgot to mention---I also have the NMR assignment(HNCACB spectrum) of the protein, is it possible to combine the NMR data in my refinement? Regards, On Fri, Jan 6, 2012 at 4:14 PM, 商元 shangyuan5...@gmail.com wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Hi, You are touching upon several issues here. The first question to ask is how good and complete are your data to 3.2 A resolution. This should be your first concern. Are they the best you can get at this stage? Second, you're absolutely correct in that there is a lot more to do to improve your model. Although it sounds as if you're on the right track, having the Rw/Rf values so close sounds alarming. Also, with low resolution data you can expect high B's but of course you should try your best to fit residues/atoms to the e.d. Setting high weights to the geometry/stereochemistry restraints in whichever refinement program you're using, can help here too, at least in the initial stages of the refinement if not throughout. If you're using the automatic weights offered by the program you may want to examine them carefully and see whether they can be modified to improve your convergence. So you have a lot to do before considering throwing in your NMR data. As for the latter, I think that there have been a few papers recently from David Baker's lab and Guy Montelione's lab where they've shown how to use rudimentary NMR data (i.e. before converting them to NOE's) in refinement of crystal structures. Which brings up the next question: are you not going to calculate NOE's from your data ? Are the NMR data that you have not sufficient to derive a good solution structure? NOE distance restraints on their own can be used to improve crystallographic structures (I can send you some old and recent references off list, if you're interested). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 商元 [shangyuan5...@gmail.com] Sent: Friday, January 06, 2012 10:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Structure Determination combining X-ray Data and NMR Also, there is one more information I forgot to mention---I also have the NMR assignment(HNCACB spectrum) of the protein, is it possible to combine the NMR data in my refinement? Regards, On Fri, Jan 6, 2012 at 4:14 PM, 商元 shangyuan5...@gmail.com wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assumingoccupancyis 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set areasonable B-factor values in the refinement? Best Regards, Yuan
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Hi Yuan, Bad geometry is a general issue for most low resolution structure refinement. There are quite a lot papers discussing it. I think you can try to set a reference structure or set high restrain in refinement, which should be easily achieved in Phenix. How did you know the B-factors are too high? There is no standard for a single case. B-factors varies from crystal to crystal even them have the same molecular content. Wilson-B factor may be a good indicator. I am a little bit worried about your R and R-free. Them are too close (Rfree is even lower than R!). What is your space group? If you want to reduce refining parameters to increase data/para ratio, you can try group B-factor refinement in Phenix. For combining NMR data, you should have NOE assignment. Best, Zhiyi On 1/6/12, 商元 shangyuan5...@gmail.com wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
and R-factor/R-free have a value of 0.328/0.326. Notice that RfreeR. This may be caused by twinning and/or NCS, as the test set is not truly independent of the working set. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Normally, at 3.2A you would have strong geometry restraints, thus reducing the effective number of degrees of freedom to perhaps as few as two per residue. Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? Not sure what you mean by this. Given the resolution and overall B values, it is likely that you won't have strong enough electron density to place many side chains. In which case you should omit the atoms unsupported by electron density from your model. As a general rule, at 3.2A you should be able to trace the backbone and place some sidechains. How to set a reasonable B-factor values in the refinement? The B-factor values are what they are, you cannot set them to a reasonable value of your choice. It is not entirely unusual to see B~100 at 3.2A, so don't worry too much about that. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
As others have mentioned, your geometry/x-ray weight may need to be adjusted. However, at 3.2 Angstroms I'd recommend against using atomic B-factors - the rule of thumb for this is 2.8 Angstroms for atomic B-factors (or at least it was back in the day). It might help to use an overall B-factor combined with one (or a few) TLS groups. Regarding how far to trust the density from the refined model - that's what (composite) omit maps are for. Good luck, Pete 商元 wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
2012/1/6 Pete Meyer pame...@mcw.edu: However, at 3.2 Angstroms I'd recommend against using atomic B-factors - the rule of thumb for this is 2.8 Angstroms for atomic B-factors (or at least it was back in the day). It might help to use an overall B-factor combined with one (or a few) TLS groups. This may be true for older software which restraints B-factors only to bonded atoms, but it is not the case in Phenix*, which takes into account all nearby atoms, not just bonded ones. The result is that individual B-factor refinement is very stable at low resolution - we don't know what the limit is, but it routinely works very well at 4A. Of course the performance is still dependent on solvent content, NCS, etc., but it is very rare that grouped B-factor refinement actually works better. -Nat * I think Refmac may do something similar, but I haven't tried this recently. I would be very surprised if it did not work well at 3.2A, however.
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
First thing I would try to shoot more crystals. Easy way out. I once struggled with a 2.7A data set for weeks only to find out I had a 1.5A diffracting crystal taking a bath in some storage buffer right next to my bench. You mention you have at this point you are looking at 25% rotamer outliers. I wonder if at 3.2A if you really have density for these side chains. You may be trying to fit these side chains in very weak unreliable (e.g. noise) electron density. As Ed Pozharski suggested, omitting these may be the right thing to do. How certain are you of your space group and how did you generate you Rfree test set. You should have RworkRfree after refining your model. HTH Yuri
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
correction: You should NOT have RworkRfree
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
This may be true for older software which restraints B-factors only to bonded atoms, but it is not the case in Phenix*, which takes into account all nearby atoms, not just bonded ones. The result is that individual B-factor refinement is very stable at low resolution - we don't know what the limit is, but it routinely works very well at 4A. B-factor refinement being stable is one thing; quieting my paranoia regarding over-fitting at low resolutions is another. Thanks for pointing this out to me - I'll have to check out the details of how phenix handles it, and give it a try. Pete
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
On Fri, Jan 6, 2012 at 10:00 AM, Pete Meyer pame...@mcw.edu wrote: Thanks for pointing this out to me - I'll have to check out the details of how phenix handles it, and give it a try. Details can be found here: http://www.phenix-online.org/presentations/latest/pavel_refinement_general.pdf (page 61 and around) and here: http://www.phenix-online.org/newsletter/ (see article On atomic displacement parameters...). Pavel
[ccp4bb] At what resolution is (individual,group,one per residue, two per residue) appropriate? was Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Pete brings out two concerns: [1] B-factor refinement being stable, [2] over-fitting at low resolutions. Here I'll suggest low resolution to be in the 3-4A range. I've seen the following question asked: At what resolution is (individual,group,one per residue, two per residue,overall) appropriate? One common response is try a number of different B-factor refinement protocols, use Rfree as a guide to determine which one is appropriate. I guess this is along the lines of B-factor refinement being stable. Seems reasonable to me. However is this approach sufficient justification to address over-fitting at low resolution? I'd welcome any thoughts on this ... F On Jan 6, 2012, at 11:00 AM, Pete Meyer wrote: B-factor refinement being stable is one thing; quieting my paranoia regarding over-fitting at low resolutions is another. Thanks for pointing this out to me - I'll have to check out the details of how phenix handles it, and give it a try. Pete - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] At what resolution is (individual,group,one per residue, two per residue) appropriate? was Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
On Fri, 2012-01-06 at 11:18 -0700, Francis E Reyes wrote: I've seen the following question asked: At what resolution is (individual,group,one per residue, two per residue,overall) appropriate? My personal opinion is that the individual B-factor refinement with restraints proper to the resolution is the only thing you will ever need. This thread may be interesting to you. http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg14133.html One common response is try a number of different B-factor refinement protocols, use Rfree as a guide to determine which one is appropriate. I guess this is along the lines of B-factor refinement being stable. Seems reasonable to me. However is this approach sufficient justification to address over-fitting at low resolution? As a general rule, the R-free should never be the only criterion. Additional criteria will vary depending on what alternative protocols you are investigating. To try to generalize, I'd say that improved unbiased fit (via R-free) should be combined with the requirement that model parameters are physically meaningful. For example, the two-per-residue B-factor model fails miserably on that account, and my sincere wish is that it's use in modern refinement software will be obfuscated to the extent needed to render it unusable. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] At what resolution is (individual,group,one per residue, two per residue) appropriate? was Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Francis, One common response is try a number of different B-factor refinement protocols, use Rfree as a guide to determine which one is appropriate. I recognize my advice along those lines above -:) Although now I would say (assuming phenix.refine): Try individual ADP refinement first, and if it fails then try group ADP refinement. By fails I mean overfitting as judged by Rfree and Rfree-Rwork. I guess this is along the lines of B-factor refinement being stable. I don't know what that means : B-factor refinement being stable. However is this approach sufficient justification to address over-fitting at low resolution? I think yes. Pavel
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
On Friday, January 06, 2012 09:30:22 am Nat Echols wrote: 2012/1/6 Pete Meyer pame...@mcw.edu: However, at 3.2 Angstroms I'd recommend against using atomic B-factors - the rule of thumb for this is 2.8 Angstroms for atomic B-factors (or at least it was back in the day). �It might help to use an overall B-factor combined with one (or a few) TLS groups. This may be true for older software which restraints B-factors only to bonded atoms, but it is not the case in Phenix*, which takes into account all nearby atoms, not just bonded ones. The result is that individual B-factor refinement is very stable at low resolution - we don't know what the limit is, but it routinely works very well at 4A. Unfortunately, stable and statistically correct are two very different criteria. It is quite possible to have a stable refinement that produces nonsensical, or at least unjustifiable, B factors. Actually this caveat applies to things other than B factors as well, but I'll stay on topic. At last year's CCP4 Study Weekend I presented a statistical approach to deciding what treatment of B could be justified at various resolutions. To B or not to B? The presentations from that meeting should appear in a special issue of Acta D soon. Based on the set of representative cases I have examined, I am willing to bet that with the limited obs/parameter ratio in the case at hand, a model with individual Bs would turn out to be statistically unjustified even if the refinement is stable. A TLS model is more likely to be appropriate. cheers, Ethan Of course the performance is still dependent on solvent content, NCS, etc., but it is very rare that grouped B-factor refinement actually works better. -Nat * I think Refmac may do something similar, but I haven't tried this recently. I would be very surprised if it did not work well at 3.2A, however. -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
On Fri, 2012-01-06 at 10:48 -0800, Ethan Merritt wrote: A TLS model is more likely to be appropriate. A quick clarification request if I may: We all seen how well the multi-group TLS models seem to match the B-factor variation along the chain. Is this in your opinion how such model may be really effective, by incorporating most of the B-factor variation into ~100 TLS parameters? And a question: Given that the B-factors for side chain atoms will be generally higher, do you know if creating two separate sets of TLS parameters for backbone / side chains improves things? Thanks, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
On Friday, January 06, 2012 11:15:11 am Ed Pozharski wrote: On Fri, 2012-01-06 at 10:48 -0800, Ethan Merritt wrote: A TLS model is more likely to be appropriate. A quick clarification request if I may: We all seen how well the multi-group TLS models seem to match the B-factor variation along the chain. Is this in your opinion how such model may be really effective, by incorporating most of the B-factor variation into ~100 TLS parameters? I have run statistical analysis of alternative models on various structures in the resolution range 2.8 - 4.0 A. For some of these, I found that a full 1-Biso-per-atom model was indeed statistically justified. For most, however, a TLS model was better. For some, a hybrid Biso + TLS model was better than either alone. So this really should be decided on a case by case basis rather than trying to come up with a single rule of thumb. Now as to how many TLS groups a model should be partitioned into, that varies all over the place and is clearly a consequence of the individual lattice packing. For some structures with loose packing (as I interpret the cause), a single-group TLS model with uniform constant per-atom B is significantly better than a model with a separate B factor for each atom but no TLS component. Adding additional TLS groups does not actually help that much. To me this means that the largest factor contributing to the ADPs is the overall displacement of the whole molecule within the lattice, which is strongly anisotropic. The single-group TLS model describes this anisotropy well enough, while any number of isotropic B factors does not. Those cases where the individual B factor option tests out best correspond, as I interpret it, to relatively rigid lattice packing. In these crystals the overall anisotropy is very low, so TLS models are not the right formalism to use in describing the distribution of ADPs. Perhaps normal-mode models would be better; it is hard to draw conclusions from the very small number of normal-mode refinements reported to date. And a question: Given that the B-factors for side chain atoms will be generally higher, do you know if creating two separate sets of TLS parameters for backbone / side chains improves things? That is a question that I am currently working on. I don't think that two sets of TLS parameters will turn out to be a good way to handle it. I am more attracted to the idea of applying a TLS description on top of a fixed a priori model for B variation along the sidechain. This approach is inspired by the per-amino acid targets for varying B along the sidechain that were developed by Dale Tronrud for use in TNT. cheers, Ethan Thanks, Ed. -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
