Re: [ccp4bb] model bias
And given that all these maps (Fo, Fc, and their differences) are made without the F000 term and so have average value zero, it is highly likely that Fc will be negative in the region of the deleted subunit (solvent is the lowest density in the model except for the chinks of vacuum between the atoms in the protein, and averaging over the volume of the protein you expect protein to be electron-denser than solvent). If Fc is negative then Fo-Fc negative implies Fo is still more negative, and 2fo-fc cannot be positive. It would be instructive to make Fo and Fc maps and check their values in the region of the deleted subunit to resolve the contradiction. On 10/11/2017 04:47 PM, Edward A. Berry wrote: This is hard to understand/impossible, at least in terms of simple 2fo-fc and fo-fc maps. We can think of a difference map as the difference between two maps, like fo-fc map = fo map - fc map. There is no model in the region, since you deleted it, so Fc=0, and Fo-Fc negative => Fo<0, so 2fo-fc cannot be positive. No, wait, there will be solvent, so Fc = Rho(solvent) Fo-Fc is negative, so Fo 2Fo>Rho(solv) 2Fo > Fc > Fo Fo > 1/2 Fc So this would be possible if the density of your protein is less than that of the solvent but more than half that of the solvent. Partial occupancy? but where the protein is missing there would be solvent (in Fo if not in Fc), so you still couldn't get below density of the solvent. Maybe its a problem with the solvent model? On 10/11/2017 09:48 AM, Karsten Dreifus wrote: Dear all, I have a 120 aa protein. Matthews coefficient indicates 3 mol in asu. Molrep (template with 70 % seq identity) finds three NCS molecules (the template model has only 1 chain as it is in different space group). Now, REFMAC refines it to 25/30 % R/RFREE. I was just worried about model bias, so I just deleted chain C and re-ran the refinement. I observe that in the place of chain C, the DELFWT map in Coot shows negative density (red colour). Shouldnt the deleted regions show POSITIVE density (green colour)? The 2fo-fc map in the deleted region shows usual blue colour. I shook the 2chain model with Phenix simple dynamics and then ran refinement with same results. How do I verify the model is correct? Karsten
Re: [ccp4bb] model bias
This is hard to understand/impossible, at least in terms of simple 2fo-fc and fo-fc maps. We can think of a difference map as the difference between two maps, like fo-fc map = fo map - fc map. There is no model in the region, since you deleted it, so Fc=0, and Fo-Fc negative => Fo<0, so 2fo-fc cannot be positive. No, wait, there will be solvent, so Fc = Rho(solvent) Fo-Fc is negative, so Fo 2Fo>Rho(solv) 2Fo > Fc > Fo Fo > 1/2 Fc So this would be possible if the density of your protein is less than that of the solvent but more than half that of the solvent. Partial occupancy? but where the protein is missing there would be solvent (in Fo if not in Fc), so you still couldn't get below density of the solvent. Maybe its a problem with the solvent model? On 10/11/2017 09:48 AM, Karsten Dreifus wrote: Dear all, I have a 120 aa protein. Matthews coefficient indicates 3 mol in asu. Molrep (template with 70 % seq identity) finds three NCS molecules (the template model has only 1 chain as it is in different space group). Now, REFMAC refines it to 25/30 % R/RFREE. I was just worried about model bias, so I just deleted chain C and re-ran the refinement. I observe that in the place of chain C, the DELFWT map in Coot shows negative density (red colour). Shouldnt the deleted regions show POSITIVE density (green colour)? The 2fo-fc map in the deleted region shows usual blue colour. I shook the 2chain model with Phenix simple dynamics and then ran refinement with same results. How do I verify the model is correct? Karsten
Re: [ccp4bb] model bias
A round of refinement with simulated annealing followed by minimization should address your concern. Pavel On Wed, Oct 11, 2017 at 4:48 PM, Karsten Dreifus wrote: > Dear all, > I have a 120 aa protein. Matthews coefficient indicates 3 mol in asu. > Molrep (template with 70 % seq identity) finds three NCS molecules > (the template model has only 1 chain as it is in different space > group). Now, REFMAC refines it to 25/30 % R/RFREE. I was just worried > about model bias, so I just deleted chain C and re-ran the refinement. > I observe that in the place of chain C, the DELFWT map in Coot shows > negative density (red colour). Shouldnt the deleted regions show > POSITIVE density (green colour)? The 2fo-fc map in the deleted region > shows usual blue colour. I shook the 2chain model with Phenix simple > dynamics and then ran refinement with same results. > > How do I verify the model is correct? > Karsten >
Re: [ccp4bb] model bias
Dear All, thank you all for your suggestions and support. The quick look (by removing interacting AA and subsequent refinement with and without little SA) revealed that some AAs are mislocated and indeed seem to be more directed to the density belonging to the ligand. So, I will try "some" of your sugestions to intervene bias directly after MR before further refinement. Thank you again! Regards, Aleks -- --- Aleksandar Bijelic, MSc. Institut für Biophysikalische Chemie Universität Wien Althanstrasse 14 A-1090 Wien Tel: +43 1 4277 52536 e-Mail: aleksandar.bije...@univie.ac.at
Re: [ccp4bb] model bias
Dear Aleksandar, I did use SA-OMIT map feature in Phenix, implemented by Tom Terwilliger. I would strongly recommend you to use this feature. Remove the suspicious residues from the pdb and run first SA-omit map in phenix with default settings, and later with different starting temperatures. Make sure also to use harmonic restrains to prohibit neighboring residues to creep into the "omitted" region. Also, use MR solution and the raw mtz file with only FP and SIGFP that you have for the omit map. I have experienced that if you use refined model then it does not completely get rid of biases. At max, you can use rigid body refined one. On the other side, if your molecule is not too big, then you can try the CNS. In that case, I would recommend remove the 'suspicious' resiudes and 1st use segmented rigid body refinement, i.e, consider each chain or subunit as rigid entity. Then, you can try group B factor with restrains along with DEN mode for simulated annealing. I strongly recommend the DEN mode which can help you to remove bias. Please note that it is necessary to try out several different temperatures and different temperature gradient as well for simulated annealing in order to reproduce the features. Otherwise, you never know if you are stuck at local minima or not. thanks shibom On Wed, Apr 29, 2015 at 4:16 AM, Aleksandar Bijelic < aleksandar.bije...@univie.ac.at> wrote: > Dear CCP4 users, > > I am currently solving a structure (2.8-2.9 A resolution) of a protein > complexed with a ligand using MR with the apo-form of this protein as model > (resolution of the model is 2.4). After MR-phasing I performed a regular > autobuild run giving me good outputs and thus I refined the best pdb > leading to good values according to R-values and geometry, however, the > denstiy doesn´t look well (but I think it´s due to the moderate > resolution). Now I want to get sure if the side chains which are involved > in the ligand binding are correctly positioned. However, the active site is > suspicously similar to the active site of the model (apo-form) and so I am > afraid that this could be due to model bias. My question is how to check > and to get rid of the bias (if present) at this stage (after several > refinements). I read the publication of Terwilliger about iterative-build > OMIT maps but since I am a bloody novice in this field I didn´t really > understand it. I originally thought iterative-build OMIT maps are performed > to compare the output map with one´s map in order to detect uncertainties, > but what to do next? Or should I start from the beginning but how to > proceed than, what should I do (I am using Phenix via GUI) ... Is it > possible and reasonable to run autobuild with iterative omit map option? Or > is it only reasonable if experimental phases are available? I didn´t run > iterative-build OMIT maps yet because I am not sure how to run it correctly > (what method is the best?) and at my institute the run will take more than > 1 day and I don´t want to block one computer until I am not sure if it is > reasonable. I hope you can give me some advice and help me. Thank you in > advance. > > Regards, > > Aleks > > -- > --- > Aleksandar Bijelic, MSc. > > Institut für Biophysikalische Chemie > Universität Wien > Althanstrasse 14 > A-1090 Wien > > Tel: +43 1 4277 52536 > e-Mail: aleksandar.bije...@univie.ac.at > > >
Re: [ccp4bb] model bias
Hi Aleks: Maybe you can try CNS ( Initial refinement by simulated annealing) also. It may help to get rid of the model bias and takes short time to run. Xiaodi > Date: Wed, 29 Apr 2015 13:52:53 +0200 > From: frederic.velli...@ibs.fr > Subject: Re: [ccp4bb] model bias > To: CCP4BB@JISCMAIL.AC.UK > > Hello, > > I would certainly try the "usual approaches" (map coefficients that are > less sensitive to model-bias, i.e. Sigmaa; OMIT maps - there are several > of these which can be calculated). In addition, you may have a look at > the approach of Liu and Xiong (2014, J. Mol. Biol. 426, 980-993). It is > a well known technique (applying a negative temperature factor to > amplitudes, for map sharpening) but this paper describes a systematic > study indicating improvement whatever the situation. > > As usual in macromolecular crystallography, confidence is gained by the > use of several approaches. > > HTH, > > Fred. > > On 29/04/15 13:16, Aleksandar Bijelic wrote: > > Dear CCP4 users, > > > > I am currently solving a structure (2.8-2.9 A resolution) of a protein > > complexed with a ligand using MR with the apo-form of this protein as > > model (resolution of the model is 2.4). After MR-phasing I performed a > > regular autobuild run giving me good outputs and thus I refined the > > best pdb leading to good values according to R-values and geometry, > > however, the denstiy doesn´t look well (but I think it´s due to the > > moderate resolution). Now I want to get sure if the side chains which > > are involved in the ligand binding are correctly positioned. However, > > the active site is suspicously similar to the active site of the model > > (apo-form) and so I am afraid that this could be due to model bias. > > My question is how to check and to get rid of the bias (if present) at > > this stage (after several refinements). I read the publication of > > Terwilliger about iterative-build OMIT maps but since I am a bloody > > novice in this field I didn´t really understand it. I originally > > thought iterative-build OMIT maps are performed to compare the output > > map with one´s map in order to detect uncertainties, but what to do > > next? Or should I start from the beginning but how to proceed than, > > what should I do (I am using Phenix via GUI) ... Is it possible and > > reasonable to run autobuild with iterative omit map option? Or is it > > only reasonable if experimental phases are available? I didn´t run > > iterative-build OMIT maps yet because I am not sure how to run it > > correctly (what method is the best?) and at my institute the run will > > take more than 1 day and I don´t want to block one computer until I am > > not sure if it is reasonable. I hope you can give me some advice and > > help me. Thank you in advance. > > > > Regards, > > > > Aleks > > > > > -- > Fred. Vellieux (B.Sc., Ph.D., hdr) > > IBS / ELMA > Campus EPN > 71 avenue des Martyrs > CS 10090 > F-38044 Grenoble Cedex 9 > Tel: +33 457428605 > Fax: +33 476501890
Re: [ccp4bb] model bias
Hello, I would certainly try the "usual approaches" (map coefficients that are less sensitive to model-bias, i.e. Sigmaa; OMIT maps - there are several of these which can be calculated). In addition, you may have a look at the approach of Liu and Xiong (2014, J. Mol. Biol. 426, 980-993). It is a well known technique (applying a negative temperature factor to amplitudes, for map sharpening) but this paper describes a systematic study indicating improvement whatever the situation. As usual in macromolecular crystallography, confidence is gained by the use of several approaches. HTH, Fred. On 29/04/15 13:16, Aleksandar Bijelic wrote: Dear CCP4 users, I am currently solving a structure (2.8-2.9 A resolution) of a protein complexed with a ligand using MR with the apo-form of this protein as model (resolution of the model is 2.4). After MR-phasing I performed a regular autobuild run giving me good outputs and thus I refined the best pdb leading to good values according to R-values and geometry, however, the denstiy doesn´t look well (but I think it´s due to the moderate resolution). Now I want to get sure if the side chains which are involved in the ligand binding are correctly positioned. However, the active site is suspicously similar to the active site of the model (apo-form) and so I am afraid that this could be due to model bias. My question is how to check and to get rid of the bias (if present) at this stage (after several refinements). I read the publication of Terwilliger about iterative-build OMIT maps but since I am a bloody novice in this field I didn´t really understand it. I originally thought iterative-build OMIT maps are performed to compare the output map with one´s map in order to detect uncertainties, but what to do next? Or should I start from the beginning but how to proceed than, what should I do (I am using Phenix via GUI) ... Is it possible and reasonable to run autobuild with iterative omit map option? Or is it only reasonable if experimental phases are available? I didn´t run iterative-build OMIT maps yet because I am not sure how to run it correctly (what method is the best?) and at my institute the run will take more than 1 day and I don´t want to block one computer until I am not sure if it is reasonable. I hope you can give me some advice and help me. Thank you in advance. Regards, Aleks -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS / ELMA Campus EPN 71 avenue des Martyrs CS 10090 F-38044 Grenoble Cedex 9 Tel: +33 457428605 Fax: +33 476501890
Re: [ccp4bb] model bias
If you know what residues are likely to be involved, then set their occupancies to 0.0 (In coot go to Measures - Residue info - edit occ) then do a few cycles of refinement with that model, and then see if there is difference density for those residues... Quicker than an omit map procedure. Eleanor On 29 April 2015 at 12:16, Aleksandar Bijelic < aleksandar.bije...@univie.ac.at> wrote: > Dear CCP4 users, > > I am currently solving a structure (2.8-2.9 A resolution) of a protein > complexed with a ligand using MR with the apo-form of this protein as model > (resolution of the model is 2.4). After MR-phasing I performed a regular > autobuild run giving me good outputs and thus I refined the best pdb > leading to good values according to R-values and geometry, however, the > denstiy doesn´t look well (but I think it´s due to the moderate > resolution). Now I want to get sure if the side chains which are involved > in the ligand binding are correctly positioned. However, the active site is > suspicously similar to the active site of the model (apo-form) and so I am > afraid that this could be due to model bias. My question is how to check > and to get rid of the bias (if present) at this stage (after several > refinements). I read the publication of Terwilliger about iterative-build > OMIT maps but since I am a bloody novice in this field I didn´t really > understand it. I originally thought iterative-build OMIT maps are performed > to compare the output map with one´s map in order to detect uncertainties, > but what to do next? Or should I start from the beginning but how to > proceed than, what should I do (I am using Phenix via GUI) ... Is it > possible and reasonable to run autobuild with iterative omit map option? Or > is it only reasonable if experimental phases are available? I didn´t run > iterative-build OMIT maps yet because I am not sure how to run it correctly > (what method is the best?) and at my institute the run will take more than > 1 day and I don´t want to block one computer until I am not sure if it is > reasonable. I hope you can give me some advice and help me. Thank you in > advance. > > Regards, > > Aleks > > -- > --- > Aleksandar Bijelic, MSc. > > Institut für Biophysikalische Chemie > Universität Wien > Althanstrasse 14 > A-1090 Wien > > Tel: +43 1 4277 52536 > e-Mail: aleksandar.bije...@univie.ac.at > > >
Re: [ccp4bb] model bias
Hi Manoj, Following on from Poul's reply, and maybe whilst you are waiting to get derivatives ;) you could try something like the following for getting around the model-bias-after-borderline-MR-issue. 1) generate a prime-&-switch'd map from resolve. 2) use this map to prune your model (be quite brutal here). 3) Re-refine, using refmac and, in parallel, a simulated annealing refinement protocol (CNS/Phenix). 4) Stick your pruned models and all these maps into coot and see what you can build back. I've found that schemes/strategies based around this can break the back of refinement after borderline MR. HTH Dave 2009/8/11 ManojSaxena : > Hi all, > > I am working with a protein that have 28% similar to my MR template. > I have processed data in HKL2000 for one of my crystal and I got unique sol > in space group > P212121. with LLG 131 and TFZ score 13.5 > I have used buccaneer and coot for model building and my Rfee came to 45%. > I used the PDB file from this crystal ( Rfee of 45%) as my MR model for data > obtained from > another crystal and got sol with TFZ score=40.5 and LLG=2305. > > I used coot and did a round of refmac refinement now my Rfee is 41%. > > My concern is > a) wether my scheme is good or not because I am afraid that this will > increase the model > bias. > b)Now I am stuck at 41% Rfee I have many chain breaks and loop regions have > not good > density. I tried TLS refinement it did not help. what else I shall try?? > > Thanks > > Manoj > PSU > Biochemistry > -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB
Re: [ccp4bb] model bias
You dont give the resolution of your data. There are always things to check 1) space group - could it be P222 or P21 2 2 or P 21 21 2 or P2 21 2 etc etc - there are 8 possibilities. You can use absences along the axial lines to help decide on the screw axes, but these can be misleading if you have more than 1 molecule in the asymmetric unit and the molecules are related by a non-crrystallographic translation. Phaser can be run in each space group in turn and usually that can decide on SG. Did you do that? 2) Is there a problem with the data - severe anisotropy? twinning? etc etc 3) Maybe you just need to do more rebuilding - depending on theresolution some of the automated procedures can help - Arp/wARP, buccaneer and so on.. Eleanor ManojSaxena wrote: Hi all, I am working with a protein that have 28% similar to my MR template. I have processed data in HKL2000 for one of my crystal and I got unique sol in space group P212121. with LLG 131 and TFZ score 13.5 I have used buccaneer and coot for model building and my Rfee came to 45%. I used the PDB file from this crystal ( Rfee of 45%) as my MR model for data obtained from another crystal and got sol with TFZ score=40.5 and LLG=2305. I used coot and did a round of refmac refinement now my Rfee is 41%. My concern is a) wether my scheme is good or not because I am afraid that this will increase the model bias. b)Now I am stuck at 41% Rfee I have many chain breaks and loop regions have not good density. I tried TLS refinement it did not help. what else I shall try?? Thanks Manoj PSU Biochemistry
Re: [ccp4bb] model bias
Great that you have MR phases - it will help you identify heavy-atom sites for phasing and perhaps even the sulphur sites will be enough. Poul On 11/08/2009, at 20.33, ManojSaxena wrote: Hi all, I am working with a protein that have 28% similar to my MR template. I have processed data in HKL2000 for one of my crystal and I got unique sol in space group P212121. with LLG 131 and TFZ score 13.5 I have used buccaneer and coot for model building and my Rfee came to 45%. I used the PDB file from this crystal ( Rfee of 45%) as my MR model for data obtained from another crystal and got sol with TFZ score=40.5 and LLG=2305. I used coot and did a round of refmac refinement now my Rfee is 41%. My concern is a) wether my scheme is good or not because I am afraid that this will increase the model bias. b)Now I am stuck at 41% Rfee I have many chain breaks and loop regions have not good density. I tried TLS refinement it did not help. what else I shall try?? Thanks Manoj PSU Biochemistry
Re: [ccp4bb] model bias after MR and rigid body
Presumably you only want to see if the mutation has been successful, and check for other gross changes? It would not be sensible to try to get a well refined model from such low resolution, espec if there is already a structure M" with better data.. So the problem of model bias in refinement is not so serious. I would merge the M2 data with the new mutants - get FCs for the M2 structure, perhaps after removing those residues you have mutated and cutting back some side chains to see how well your new data produces the missing density.. Then do Fmut - Fc maps, and maybe also Fo(mut) - fobs(M2) difference maps with PHIC and FOM. You should see large holes or peaks depending on the changes you have done which should answer your questions. Eleanor [EMAIL PROTECTED] wrote: Hi All: There are two published x-ray structures (very similar, RMSD<1A if ignore 2 loops) available for a wild-type protein; let's call these two structures M1 and M2. They belong to the same space group but with large differences in unit cell dimensions (over 10%, or 10A in each dimension, cross R>30%). Now I have some (deletion) mutant structures to solve and the unit cell dimensions of these mutant crystals are very similar to one of the crystal forms (say M2)mentioned above. All these crystals including the wild-type diffract to low resolution (~3.5-4A) and have no NCS, so the parameter/data ratio must be very high. My question is: would model bias be less if I use the structure model that is very different (i.e M1) in unit cell dimensions? In other words, if I only do molecular replacement and rigid body refinement, and look at the 2Fo-Fc and Fo-Fc maps right after this, will I see a less biased map by using M1 as the model instead of M2? In my naive thinking, since the M1 and M2 crystal forms are so different, and the parameters in the M1 model has essentially not been refined against the M2 data (which the mutant belong to). Using M1 as the model to calculate maps must be different from using M2, and presumably, with less model bias towards the wild type structure? In contrast, if the M2 model is used for these mutants. since all parameters of M2 has been fully refined against similar data in wild-type, all these parameters might presumably have been "contaminated" by the wild-type data? i.e. all the parameters (all parts of the wt model) have been adjusted "together" to fit the data. If I use this model on a very similar mutant data, will it be more likely to generate back the wt model(more biased)? Do you think this is a valid argument? Regards, Weikai
