[ccp4bb] ctruncate bug?
Hi I seem to be getting a lot of outliers rejected by Phaser with data processed with the latest ctruncate which are not present when data is processed with the older version (or old truncate) - has something been changed in the code that would cause this? With CCP4 6.4: ctruncate version 1.15.9 : 15/07/14 Phaser logfile: Outliers with a probability less than 1e-06 will be rejected There were 489 (0.5620%) reflections rejected HKL resoF probability 94 49 1.65 126.969 8.862e-41 -25 50 1.66 124.322 2.404e-30 05 50 1.66 131.982 3.516e-35 -147 47 1.66 123.548 3.541e-27 20 20 35 1.66 87.870 5.191e-88 -1 15 46 1.66 114.059 8.326e-130 -6 21 41 1.67 104.887 7.047e-50 -94 49 1.67 120.765 5.821e-22 3 34 20 1.67 93.244 6.844e-41 74 49 1.67 131.702 2.985e-36 -3 34 20 1.67 106.603 5.786e-57 4 370 1.67 119.036 3.934e-48 1 16 45 1.67 107.599 2.356e-55 -149 46 1.67 122.033 2.371e-24 -5 33 22 1.67 110.802 3.492e-60 3 33 22 1.68 102.663 6.189e-46 -74 49 1.68 120.855 1.875e-20 -26 49 1.68 132.650 6.233e-84 9 19 41 1.68 91.717 2.857e-21 -17 12 43 1.69 112.959 5.298e-37 More than 20 outliers (see VERBOSE output) With CCP4 6.4: ctruncate version 1.15.5 : 05/06/14 Phaser logfile: Outliers with a probability less than 1e-06 will be rejected There were 1 (0.0011%) reflections rejected HKL resoF probability 020 31.00 913.757 1.369e-08 Thanks, Huw
Re: [ccp4bb] dynapro DLS cuvettes
Here's another one, catalog number 105-250-15-40: http://www.hellma-analytics.com/kuevetten/136/en/pg_id,41$g_id,24$item_id,63/fluorescence-cells.html#63 We use that for light scattering on a Zetasizer Nano. Andreas On 14/08/2014 10:35, Gloria Borgstahl wrote: Does any one know of a source of these cuvettes? Protein Solution doesn't exist anymore and Wyatt no longer has these. -- Andreas Förster Crystallization and X-ray Facility Manager Centre for Structural Biology Imperial College London
[ccp4bb] cc1/2 value in hkl2000
Hello everyone Can anybody please tell me where to locate the Corelation value between half sets (CC1/2) of a data processed through HKL2000 ?? -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cc1/2 value in hkl2000
Probably the only way is to take unmerged scalepack output to aimless. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Faisal Tarique faisaltari...@gmail.com /divdivDate:08/15/2014 9:40 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] cc1/2 value in hkl2000 /divdiv /divHello everyone Can anybody please tell me where to locate the Corelation value between half sets (CC1/2) of a data processed through HKL2000 ?? -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cc1/2 value in hkl2000
On 08/15/2014 09:40 AM, Faisal Tarique wrote: Hello everyone Can anybody please tell me where to locate the Corelation value between half sets (CC1/2) of a data processed through HKL2000 ?? First of all you must have a recent version of the suite- it didn't exist in earlier versions. Then, it is in the scalepack log file, last table, with chi^2 and R-fac before the list of systematic extinctions. I presume it is also one of the plots you can display from the gui. Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac Rmeas Rpim CC1/2CC* 30.00 10.70 4410.3 209.649.3 1.489 0.056 0.064 0.062 0.026 0.996 0.999 10.70 8.56 5654.8 255.956.9 1.006 0.053 0.059 0.058 0.024 0.997 0.999 -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cc1/2 value in hkl2000
If you are using newer versions of HKL2000 it is printed in the scale.log file. On Fri, Aug 15, 2014 at 9:40 AM, Faisal Tarique faisaltari...@gmail.com wrote: Hello everyone Can anybody please tell me where to locate the Corelation value between half sets (CC1/2) of a data processed through HKL2000 ?? -- Regards Faisal School of Life Sciences JNU
[ccp4bb] FW: [ccp4bb] dynapro DLS cuvettes
Some memories from the dark recesses of my mind… Hope it helps! Bryan From: Prince, D Bryan Sent: Thursday, August 14, 2014 6:07 PM To: 'Gloria Borgstahl' Subject: RE: [ccp4bb] dynapro DLS cuvettes Dear Gloria, I seem to remember that we had a DynaPro DLS system and used disposable plastic cuvettes (low volume). I think these are the ones we used, and we were very happy with them. If memory serves, we could do about 25uL samples, and they were individually packed so cleanliness was not a concern. http://www.sigmaaldrich.com/catalog/product/sigma/z605050?lang=enregion=US Sigma has a supply of quartz cuvettes here that you might find helpful: http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=104902943 GOOD LUCK Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gloria Borgstahl Sent: Thursday, August 14, 2014 5:35 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] dynapro DLS cuvettes Does any one know of a source of these cuvettes? Protein Solution doesn't exist anymore and Wyatt no longer has these. -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] CC-half value ??
Same here. Ultimately, the KD test must be used in the end to finalize the resolution (keeping in mind recently discussed issues of effective resolution given data completeness). I just want to add that at least some versions of aimless report overestimated resolution based on CC1/2 cutoff when outliers are present (e.g. due to ice rings or salt diffraction). It seems that aimless just picks the highest resolution bin where cc1/2 0.5 even if some lower resolution bins are below 0.5 as well. I have written a script for more robust automated evaluation of these curves. In a nutshell, it fits CC1/2 (d) curve to 1/(1+exp (-x)) and returns the resolution at midpoint. I'm pretty sure that theoretical CC1/2 (d) dependence is different from this, but it seems good enough for a rough estimate. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Roger Rowlett rrowl...@colgate.edu /divdivDate:08/14/2014 5:44 PM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] CC-half value ?? /divdiv /divExactly. Aimless will give you suggested resolution cutoffs based on CC 1/2 in the log file. Roger Rowlett On Aug 14, 2014 5:04 PM, conan仙人指路 conan_...@hotmail.com wrote: Hi Faisal, CC-half standard is valuable in evaluating the cut-off of highest resolution. Sometimes even if I/sigI is close to 1 and completeness is not as high, if CC-half is still significant, it may be worth incorporate the extra high-res shell data and extend the resolution. Again, if only the reliability and unbias are carefully confirmed, and the apparent significant CC-half is not due to an artifact of some other factors like ice ring etc. (Ref: Karplus PA and Diederichs K. 2012 Science 336, 1030-1033 https://www.pubmed.com/pubmed/22628654) It has yet to be appreciated by most population of the crystallography society, unlike the I/sigI, completeness, Rsym. In particular, Rsym has gradually less a direct measurement of the data quality and or determinant of resolution cut-off. Best, Conan Hongnan Cao, Ph.D. Department of Biochemistry Rice University Date: Fri, 15 Aug 2014 01:39:48 +0530 From: faisaltari...@gmail.com Subject: [ccp4bb] CC-half value ?? To: CCP4BB@JISCMAIL.AC.UK Dear all How CC-half value of a data set determines the maximum resolution limit during data processing ?? Although much we know about the Rsym and I/Isig values of the highest resolution shell while processing the data, what are the parameters we need to check related to CC-half values ?? -- Regards Faisal School of Life Sciences JNU
[ccp4bb] Zinc binding protein expressed from insect cells
Dear all, Sorry for the non-crystallographic question. Currently I am working on a zinc binding protein which is expressed in insect cells and may contain 4-6 zinc ions. As we know, so many zinc binding proteins can absorb the iron ions from the culture medium and the protein looks from yellow to dark red when concentrated. But when I concentrate the protein, I didn’t see the red color even in the very high concentration. I am just wondering if a zinc binding protein is expressed from insect or mammalian cells, can the zinc binding sites grab the irons instead of zinc or the zinc binding site can be empty loaded if there is not enough zinc in the culture medium? If so, do I need to include some zinc salt into the culture medium when doing expression or I can add some zinc ions when purifying? Usually, how much zinc and at which step of purification can we add the zinc into the solution when doing purification? Another question is that we know DTT can react with the heavy atoms to form the insoluble sulfide precipitates and if the zinc binding protein is purified with DTT at a final concentration of 1-5 mM, can it strip the zinc ions from the protein? I am appreciated if someone has this kind of experimental experiences and thanks in advance! Heng
Re: [ccp4bb] CC-half value ??
I should make the estimation in Aimless more robust, and curve fitting sounds like a good idea (but what function?). Outliers are a difficult problem, but anyway I think you should look at the curve and not just the number estimated. I would look at I/sigI as well, and anisotropy to decide the resolution. However, the final cutoff should probably be based on refinement, and also I don't think the exact cutoff makes a huge difference (see http://www.ncbi.nlm.nih.gov/pubmed/23793146) Phil On 15 Aug 2014, at 15:54, Ed Pozharski pozharsk...@gmail.com wrote: Same here. Ultimately, the KD test must be used in the end to finalize the resolution (keeping in mind recently discussed issues of effective resolution given data completeness). I just want to add that at least some versions of aimless report overestimated resolution based on CC1/2 cutoff when outliers are present (e.g. due to ice rings or salt diffraction). It seems that aimless just picks the highest resolution bin where cc1/2 0.5 even if some lower resolution bins are below 0.5 as well. I have written a script for more robust automated evaluation of these curves. In a nutshell, it fits CC1/2 (d) curve to 1/(1+exp (-x)) and returns the resolution at midpoint. I'm pretty sure that theoretical CC1/2 (d) dependence is different from this, but it seems good enough for a rough estimate. Sent on a Sprint Samsung Galaxy S® III Original message From: Roger Rowlett Date:08/14/2014 5:44 PM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] CC-half value ?? Exactly. Aimless will give you suggested resolution cutoffs based on CC 1/2 in the log file. Roger Rowlett On Aug 14, 2014 5:04 PM, conan仙人指路 conan_...@hotmail.com wrote: Hi Faisal, CC-half standard is valuable in evaluating the cut-off of highest resolution. Sometimes even if I/sigI is close to 1 and completeness is not as high, if CC-half is still significant, it may be worth incorporate the extra high-res shell data and extend the resolution. Again, if only the reliability and unbias are carefully confirmed, and the apparent significant CC-half is not due to an artifact of some other factors like ice ring etc. (Ref: Karplus PA and Diederichs K. 2012 Science 336, 1030-1033 https://www.pubmed.com/pubmed/22628654) It has yet to be appreciated by most population of the crystallography society, unlike the I/sigI, completeness, Rsym. In particular, Rsym has gradually less a direct measurement of the data quality and or determinant of resolution cut-off. Best, Conan Hongnan Cao, Ph.D. Department of Biochemistry Rice University Date: Fri, 15 Aug 2014 01:39:48 +0530 From: faisaltari...@gmail.com Subject: [ccp4bb] CC-half value ?? To: CCP4BB@JISCMAIL.AC.UK Dear all How CC-half value of a data set determines the maximum resolution limit during data processing ?? Although much we know about the Rsym and I/Isig values of the highest resolution shell while processing the data, what are the parameters we need to check related to CC-half values ?? -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Zinc binding protein expressed from insect cells
Hi Heng, DTT can react with metal cations such as Zinc or Iron. This is why people to tend to use little DTT or no DTT at all on metal affinity columns or replace it b-mercaptoethanol or TCEP that do not interfere. Regarding the incorporation of Zinc into the culture media. I recall that Zinc finger domain expression in E coli (for nuclear receptors dna binding domains) is classically described to be done in presence of Zinc acetate added into the culture media (around 100 microMolar I think ) this is already a lot of zinc (more might be toxic to the cells). depending on what your process of purification is. I would try to add zinc in the expression media (this has probably been descrived also for insect cells) if you use a Ni or Co chelating column I would NOT add any free zinc at this stage, you can, if you wish it, add it to eluate of your affinity column and to gel filtration buffers or ion exchange buffers. That said, I would suspect that having enough zinc around is mostly beneficial at the expression stage as the protein folding machinery is dealing with your target protein.zinc misloaded protein is likely to be unsoluble. for nuclear receptors DBDs there are protocols describing reincorporation (or even exchange) of metal ions inside the domain (maybe even starting from inclusion bodies) but the more cysteines you have the more likely it is to be difficult to get the right folded protein back. so I would rather favor a strategy trying to get as much folded protein as possible by natural means (let the cells do what we biochemists still don t know to do very well). if you add free zinc I would be careful with the concentration and the pH of your buffers. at basic pH zinc and other ions (such as Ca and Fe) form insoluble hydroxydes. if you have managed to purify some protein I would try to do some emission spectroscopy to see what ions are bound (zinc and or iron ) you may be surprised by what you will see. sorry for the lengthy response but I hope this helps. all the best, Pascal Egea On Fri, Aug 15, 2014 at 8:03 AM, Harvey Rodriguez h.rodriguez.x...@gmail.com wrote: Dear all, Sorry for the non-crystallographic question. Currently I am working on a zinc binding protein which is expressed in insect cells and may contain 4-6 zinc ions. As we know, so many zinc binding proteins can absorb the iron ions from the culture medium and the protein looks from yellow to dark red when concentrated. But when I concentrate the protein, I didn’t see the red color even in the very high concentration. I am just wondering if a zinc binding protein is expressed from insect or mammalian cells, can the zinc binding sites grab the irons instead of zinc or the zinc binding site can be empty loaded if there is not enough zinc in the culture medium? If so, do I need to include some zinc salt into the culture medium when doing expression or I can add some zinc ions when purifying? Usually, how much zinc and at which step of purification can we add the zinc into the solution when doing purification? Another question is that we know DTT can react with the heavy atoms to form the insoluble sulfide precipitates and if the zinc binding protein is purified with DTT at a final concentration of 1-5 mM, can it strip the zinc ions from the protein? I am appreciated if someone has this kind of experimental experiences and thanks in advance! Heng -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] CC-half value ??
Thank you for your valuable suggestions..it really helped me a lot.. On Fri, Aug 15, 2014 at 8:38 PM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: I should make the estimation in Aimless more robust, and curve fitting sounds like a good idea (but what function?). Outliers are a difficult problem, but anyway I think you should look at the curve and not just the number estimated. I would look at I/sigI as well, and anisotropy to decide the resolution. However, the final cutoff should probably be based on refinement, and also I don't think the exact cutoff makes a huge difference (see http://www.ncbi.nlm.nih.gov/pubmed/23793146) Phil On 15 Aug 2014, at 15:54, Ed Pozharski pozharsk...@gmail.com wrote: Same here. Ultimately, the KD test must be used in the end to finalize the resolution (keeping in mind recently discussed issues of effective resolution given data completeness). I just want to add that at least some versions of aimless report overestimated resolution based on CC1/2 cutoff when outliers are present (e.g. due to ice rings or salt diffraction). It seems that aimless just picks the highest resolution bin where cc1/2 0.5 even if some lower resolution bins are below 0.5 as well. I have written a script for more robust automated evaluation of these curves. In a nutshell, it fits CC1/2 (d) curve to 1/(1+exp (-x)) and returns the resolution at midpoint. I'm pretty sure that theoretical CC1/2 (d) dependence is different from this, but it seems good enough for a rough estimate. Sent on a Sprint Samsung Galaxy S® III Original message From: Roger Rowlett Date:08/14/2014 5:44 PM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] CC-half value ?? Exactly. Aimless will give you suggested resolution cutoffs based on CC 1/2 in the log file. Roger Rowlett On Aug 14, 2014 5:04 PM, conan仙人指路 conan_...@hotmail.com wrote: Hi Faisal, CC-half standard is valuable in evaluating the cut-off of highest resolution. Sometimes even if I/sigI is close to 1 and completeness is not as high, if CC-half is still significant, it may be worth incorporate the extra high-res shell data and extend the resolution. Again, if only the reliability and unbias are carefully confirmed, and the apparent significant CC-half is not due to an artifact of some other factors like ice ring etc. (Ref: Karplus PA and Diederichs K. 2012 Science 336, 1030-1033 https://www.pubmed.com/pubmed/22628654) It has yet to be appreciated by most population of the crystallography society, unlike the I/sigI, completeness, Rsym. In particular, Rsym has gradually less a direct measurement of the data quality and or determinant of resolution cut-off. Best, Conan Hongnan Cao, Ph.D. Department of Biochemistry Rice University Date: Fri, 15 Aug 2014 01:39:48 +0530 From: faisaltari...@gmail.com Subject: [ccp4bb] CC-half value ?? To: CCP4BB@JISCMAIL.AC.UK Dear all How CC-half value of a data set determines the maximum resolution limit during data processing ?? Although much we know about the Rsym and I/Isig values of the highest resolution shell while processing the data, what are the parameters we need to check related to CC-half values ?? -- Regards Faisal School of Life Sciences JNU -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Zinc binding protein expressed from insect cells
Harvey, Depending on the zinc-binding site, it may not bind Fe(II) at all. Zn(II) and Fe(II) have very different preferred ligand binding environments. For many zinc-metalloenzymes, substitution with Fe(II) would be difficult to impossible. In general, you will find it very difficult to make your non-defined expression medium zinc-deficient. Zinc is a very common component of complex media, and is also a very common adventitious contaminant. Ideally, you will want to include the metal ion in the expression medium so that it can be incorporated at the time of protein synthesis. In many cases, this will enhance the stability of the synthesized protein. For bacterial overexpression at very high protein levels, 10-100 uM metal ion is more than plenty. More than that is actually toxic in bacterial systems, as it may impede critical iron transport into cells. But we have found that complex media already contains more than enough zinc to populate overexpressed proteins. We only supplement when we are trying to make metallosubstituted protein, in which case we use defined zinc-deficient media and supplement with a compatible metal ion (e.g., Co(II)) at 10-100 uM maximum in bacterial systems. Even that is tricky, as we need some trace metals to populate other metalloenzymes without introducing too much in the way of zinc-containing impurities. Most zinc-metalloenzymes will be immune to metal chelation by DTT or BME, as the protein-metal binding constants will be orders of magnitude higher. (Values 10^(12) are typical.) Even EDTA is not enough for many (most?) Cys(2)His(2) or Cys(2)His(OH2) sites. It is very unlikely that 1-5 mM DTT will be able to extract Zn from a metalloenzyme binding site. (We stored a particularly unstable Cys-rich zinc-metalloprotein in 100 mM DTT(!) and 2 mM EDTA (!!) and 50% glycerol and it is stable indefinitely at -20 deg C without detectable Zn loss.) You may have to evaluate the metal-binding strength of your protein experimentally or by comparison to homologous proteins. If the binding constan is expected to be 10^(10), I don't think you need to worry too much about DTT or BME. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/15/2014 11:03 AM, Harvey Rodriguez wrote: Dear all, Sorry for the non-crystallographic question. Currently I am working on a zinc binding protein which is expressed in insect cells and may contain 4-6 zinc ions. As we know, so many zinc binding proteins can absorb the iron ions from the culture medium and the protein looks from yellow to dark red when concentrated. But when I concentrate the protein, I didn’t see the red color even in the very high concentration. I am just wondering if a zinc binding protein is expressed from insect or mammalian cells, can the zinc binding sites grab the irons instead of zinc or the zinc binding site can be empty loaded if there is not enough zinc in the culture medium? If so, do I need to include some zinc salt into the culture medium when doing expression or I can add some zinc ions when purifying? Usually, how much zinc and at which step of purification can we add the zinc into the solution when doing purification? Another question is that we know DTT can react with the heavy atoms to form the insoluble sulfide precipitates and if the zinc binding protein is purified with DTT at a final concentration of 1-5 mM, can it strip the zinc ions from the protein? I am appreciated if someone has this kind of experimental experiences and thanks in advance! Heng
Re: [ccp4bb] ctruncate bug?
Hmm - Phaser doesn't usually use such high resolution data? Surprised you are getting any stuff from resolutions higher that 2A. Whether the intensity at that resolution is meaningful would need careful inspection of the truncate logs - is the wilson plot reasonable? Are the 4th moments linear, etc etc.. Lets look on Monday Eleanor On 15 August 2014 08:14, Huw Jenkins h.t.jenk...@me.com wrote: Hi I seem to be getting a lot of outliers rejected by Phaser with data processed with the latest ctruncate which are not present when data is processed with the older version (or old truncate) - has something been changed in the code that would cause this? With CCP4 6.4: ctruncate version 1.15.9 : 15/07/14 Phaser logfile: Outliers with a probability less than 1e-06 will be rejected There were 489 (0.5620%) reflections rejected HKL resoF probability 94 49 1.65 126.969 8.862e-41 -25 50 1.66 124.322 2.404e-30 05 50 1.66 131.982 3.516e-35 -147 47 1.66 123.548 3.541e-27 20 20 35 1.66 87.870 5.191e-88 -1 15 46 1.66 114.059 8.326e-130 -6 21 41 1.67 104.887 7.047e-50 -94 49 1.67 120.765 5.821e-22 3 34 20 1.67 93.244 6.844e-41 74 49 1.67 131.702 2.985e-36 -3 34 20 1.67 106.603 5.786e-57 4 370 1.67 119.036 3.934e-48 1 16 45 1.67 107.599 2.356e-55 -149 46 1.67 122.033 2.371e-24 -5 33 22 1.67 110.802 3.492e-60 3 33 22 1.68 102.663 6.189e-46 -74 49 1.68 120.855 1.875e-20 -26 49 1.68 132.650 6.233e-84 9 19 41 1.68 91.717 2.857e-21 -17 12 43 1.69 112.959 5.298e-37 More than 20 outliers (see VERBOSE output) With CCP4 6.4: ctruncate version 1.15.5 : 05/06/14 Phaser logfile: Outliers with a probability less than 1e-06 will be rejected There were 1 (0.0011%) reflections rejected HKL resoF probability 020 31.00 913.757 1.369e-08 Thanks, Huw
Re: [ccp4bb] desiring untouched ligand
Hi all and thanks for your input. I am trying to fix a sugar chain that has consecutive glucoses and I need the angle C-O-C to be as close to 104 degrees as possible keeping alpha bonds between the sugars, so when I do a Real Space Refinement in Coot, the structure gives angles close enough to 104, and the C-O bonds are at acceptable lengths. Running refmac 5 afterwards always deviates the angles by a great amount to 145 degrees or more for most of the angles. So when I run a Real Space Refinement in Coot for the Refmac5 product, the angles readjust to what is “normal” close to 104. I have tried restrained and rigid body refinement. And I did external harmonic restraints according to suggestions below by creating a pdb file having only the sentence: external harmonic residues from 843 E to 850 E sigma 0.02”, but I still get very wide angles, any suggestion? Thanks for your help, Remie On Aug 14, 2014, at 11:53 AM, Robert Nicholls nicho...@mrc-lmb.cam.ac.uk wrote: Hi Remie, You could always generate self-restraints using one chain or a specific part of a chain using prosmart. These will ensure that the model does not move too far from its current conformation. Let me know if you want help to do this. Regards, Rob On 14 Aug 2014, at 16:11, Remie Fawaz-Touma wrote: Thank you Dr. Murshudov for the information. But please I still need help. In restrained refinement, I could not find where to enter the residues I want to restrict from moving much. I was only able to find that in rigid body refinement, so I tried it and got a higher R factor than the initial file (before refinement). Is it normal to get a higher R factor when doing rigid body refinement? if not, any suggestion regarding what could be wrong? Is there a way to restrict one chain or part of a chain from moving much while doing restrained refinement? Thank you for any input, Remie On Aug 13, 2014, at 10:48 AM, Garib Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Hi Remie, You can add harmonic restraints for parts you do not want to move too much. Instructions could be found here: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html#Harmonic Regards Garib On 13 Aug 2014, at 15:44, Remie Fawaz-Touma remiefa...@gmail.com wrote: Hi everyone, Does anyone know of a way to refine with CCP4 - Refmac5 (restrained refinement is what I do) fixing a part of the the ligand? Thank you very much for your input, Remie Dr Garib N Murshudov MRC-LMB Francis Crick Avenue Cambridge CB2 0QH UK Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
