Re: [ccp4bb] AW: [ccp4bb] High R/Rfree
Remember DNA can generate some very strong reflections along the stacking axis. But 2outliers" can mean anything - as Herman says look at the images. Aimless shows you a plot of where they are - and gives a list.. - they may be associated with ice rings? or just diffraction problems. On 14 November 2017 at 08:24, wrote: > Dear Radhika, > > > > What reason does Xtriage give for declaring the reflections to be > outliers? Too weak, too strong, other reasons? As was mentioned before, > what is the resolution of your data? In cases like this, it is always good > to have a look at the diffraction images to see if there is some problem > there like streaks, ice rings etc. > > > > Best, > > Herman > > > > *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von > *Radhika Singh > *Gesendet:* Dienstag, 14. November 2017 00:45 > *An:* CCP4BB@JISCMAIL.AC.UK > *Betreff:* [EXTERNAL] [ccp4bb] High R/Rfree > > > > Hello All, > > > > I am currently working on the structure of a DNA protein complex. The > data has been processed in space group P1 (53.042 59.527 78.526 105.24 > 98.03 106.99 P 1, Rpim 11.7%). At this stage I have almost 85% model is > complete but my R/Rfree is stuck as 26%/34%. > > > > I have some concerns and questions: > > * Xtriage says there are a large number of outliers; however no > pseudotranslational symmetry is detected by the program. What are the > other reasons for outliers? > > > > * I am trying phenix.refine for refinement with the default settings. Is > there any special setting that can help me? > > > > I would like to have some suggestions about my problem. > > > > Thanks in advance > > > > Radhika > > >
[ccp4bb] AW: [ccp4bb] High R/Rfree
Dear Radhika, What reason does Xtriage give for declaring the reflections to be outliers? Too weak, too strong, other reasons? As was mentioned before, what is the resolution of your data? In cases like this, it is always good to have a look at the diffraction images to see if there is some problem there like streaks, ice rings etc. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Radhika Singh Gesendet: Dienstag, 14. November 2017 00:45 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] High R/Rfree Hello All, I am currently working on the structure of a DNA protein complex. The data has been processed in space group P1 (53.042 59.527 78.526 105.24 98.03 106.99 P 1, Rpim 11.7%). At this stage I have almost 85% model is complete but my R/Rfree is stuck as 26%/34%. I have some concerns and questions: * Xtriage says there are a large number of outliers; however no pseudotranslational symmetry is detected by the program. What are the other reasons for outliers? * I am trying phenix.refine for refinement with the default settings. Is there any special setting that can help me? I would like to have some suggestions about my problem. Thanks in advance Radhika
Re: [ccp4bb] High R/Rfree
Hi Radhika, R-factor value is almost useless unless you know the resolution (which you did not tell us): 26% is ok for 3A resolution and is nonsense for 1A, for example. The Rfree-Rwork gap is obviously large, suggesting sub-optimal refinement strategy. Try optimizing weights, let program update (add/remove/refine) water. You can send me model and data files for further diagnostics. Pavel On Mon, Nov 13, 2017 at 3:45 PM, Radhika Singh wrote: > Hello All, > > I am currently working on the structure of a DNA protein complex. The > data has been processed in space group P1 (53.042 59.527 78.526 105.24 > 98.03 106.99 P 1, Rpim 11.7%). At this stage I have almost 85% model is > complete but my R/Rfree is stuck as 26%/34%. > > I have some concerns and questions: > * Xtriage says there are a large number of outliers; however no > pseudotranslational symmetry is detected by the program. What are the > other reasons for outliers? > > * I am trying phenix.refine for refinement with the default settings. Is > there any special setting that can help me? > > I would like to have some suggestions about my problem. > > Thanks in advance > > Radhika > >
[ccp4bb] High R/Rfree
Hello All, I am currently working on the structure of a DNA protein complex. The data has been processed in space group P1 (53.042 59.527 78.526 105.24 98.03 106.99 P 1, Rpim 11.7%). At this stage I have almost 85% model is complete but my R/Rfree is stuck as 26%/34%. I have some concerns and questions: * Xtriage says there are a large number of outliers; however no pseudotranslational symmetry is detected by the program. What are the other reasons for outliers? * I am trying phenix.refine for refinement with the default settings. Is there any special setting that can help me? I would like to have some suggestions about my problem. Thanks in advance Radhika
Re: [ccp4bb] High R/Rfree after MR
in resolution limits. > > In fact I am at the moment working on one of these cases, with extremely > > large difference in resolution limits, but relatively low anisotropy. > > Anisotropy is more a deviation from "normal" intensity falloff as a > > function of resolution. There is a thin difference/relationship between the > > two concepts that I think is worth investigating. > > > > we have performed a statistical analysis of this phenomenon and the paper > > is in revision at the moment, but if you want to know where your anisotropy > > stands in respect to all the other PDBs out there, feel free to contact me > > off list. > > You mention MR: Phaser calculates the anisotropy so you can find the value > > in the first lines of the log. > > > > Staraniso or the UCLA server are good to test if you have anisotropy. > > Staraniso has a newer way of dealing with intensity falloff and accounting > > for it. > > > > All the best > > Vincent > > > > > > > > > > On 13/10/2017 10:58, Paul Miller wrote: > >> I had a similar problem to what you describe. In my case the dataset was > >> severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were > >> stuck similar to yours but the map looked good. I was told by someone with > >> a much better appreciation of the theory than myself that the anisotropy > >> was causing the problem. > >> > >> It would be interesting to know from an expert in anisotropy e.g. the > >> creators of UCLA anisotropy server or Startaniso whether anisotropy can > >> cause this problem and whether there is any way around it. > >> > >> Cheers, Paul > >> > >> Paul Steven Miller (PhD) > >> Postdoctoral Researcher > >> University of Oxford > >> Wellcome Trust Centre for Human Genetics > >> Division of Structural Biology > >> Roosevelt Drive > >> Oxford > >> OX3 7BN > >> > >> > >> Original message > >> > >>> Date: Fri, 13 Oct 2017 10:30:22 +0200 > >>> From: CCP4 bulletin board > >>> mailto:CCP4BB@JISCMAIL.AC.UK>> (on behalf of > >>> Gianluca Cioci mailto:gianluca.ci...@gmail.com>> > >>> ) > >>> Subject: [ccp4bb] High R/Rfree after MR > >>> To: > >>> CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > >>> > >>> > >>> Dear All, > >>> I am trying to refine a structure at 3.3A. Model has > >>> 60% identity to the target. Maps look OK (for 3.3A) > >>> and rebuilding in Coot is relatively > >>> straightforward. However, after some rebuilding > >>> cycles the R factors are stuck at 0.37/0.39 > >>> (REFMAC). > >>> XTRIAGE tells me that everything is normal (no twin, > >>> 98% completeness, R=3.5% in the low resolution bin), > >>> perhaps some anisotropy is present. > >>> I have already refined 2 homologous structures at > >>> resolutions going from 3.2 to 3.8 and there were no > >>> problems (final R ~ 0.21/0.24). > >>> Any advice ? > >>> Thanks, > >>> GIA > >>> > > > > -- > > Vincent Chaptal, PhD > > Institut de Biologie et Chimie des Protéines > > Drug Resistance and Membrane Proteins Laboratory > > 7 passage du Vercors > > 69007 LYON > > FRANCE > > +33 4 37 65 29 01 > > http://www.ibcp.fr<http://www.ibcp.fr/> -- === * * * Gerard Bricogne g...@globalphasing.com<mailto:g...@globalphasing.com> * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * === UT Southwestern Medical Center The future of medicine, today.
Re: [ccp4bb] High R/Rfree after MR
I would be worried by a structure with these R values, whether or not there is anisotropy to consider. Several times when this has happened to me the spacegroup turns out to be wrong. You do not give any details of the solution but especially if there is some non-crystallographic translation you can assign the space group wrongly - get a MR solution which fits some of the amplitudes perfectly ( eg h k 2l, and the rest approximately so the maps look reasonable. Have you run MR in all possible spacegroup for your point group? Eleanor On 13 October 2017 at 22:08, Gerard Bricogne wrote: > Dear Randy, > > On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote: > > Just to add to this point. The MR algorithms in Phaser are now able > > to make better use of intensity data, which is particularly > > important when you have any very weak data. Having weak data can’t > > be avoided when you have serious anisotropy (or tNCS or a > > combination of the two). Unfortunately, if you use amplitudes that > > have been through the French & Wilson (truncate) algorithm, the real > > variation in intensity is partially masked because the posterior > > amplitude values are computed on the prior assumption that all the > > reflections in a resolution shell have the same underlying intensity > > distribution. > > Your "unfortunately" calsue may be the case with the UCLA server, > but your statement is not true about what the STARANISO server (or > STARANISO as invoked within autoPROC) does: as I already indicated in > a reply to you a few months ago in this BB, the version of TRUNCATE in > STARANISO applies the French & Wilson procedure with a prior Wilson > probability whose expectation value for the intensity is modulated by > the anisotropy of the dataset. This is clearly explained on the server > - see > >http://staraniso.globalphasing.org/staraniso_about.html#step16 > > > With best wishes, > > Gerard. > > -- > > The UCLA server actually uses Phaser under the hood — what they add is > to turn the anisotropic B-values into suggested resolution limits in the > different directions. However, I don’t think they allow you yet to submit > intensities, which would be better. > > > > Best wishes, > > > > Randy Read > > > > - > > Randy J. Read > > Department of Haematology, University of Cambridge > > Cambridge Institute for Medical ResearchTel: +44 1223 336500 > > Wellcome Trust/MRC Building Fax: +44 1223 336827 > > Hills Road > E-mail: rj...@cam.ac.uk > > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > > > > On 13 Oct 2017, at 10:29, vincent Chaptal > wrote: > > > > > > Dear Gia and Paul, > > > > > > about anisotropy, one point to keep in mind is that it is not > necessarily linked to the difference in resolution limits. > > > In fact I am at the moment working on one of these cases, with > extremely large difference in resolution limits, but relatively low > anisotropy. Anisotropy is more a deviation from "normal" intensity falloff > as a function of resolution. There is a thin difference/relationship > between the two concepts that I think is worth investigating. > > > > > > we have performed a statistical analysis of this phenomenon and the > paper is in revision at the moment, but if you want to know where your > anisotropy stands in respect to all the other PDBs out there, feel free to > contact me off list. > > > You mention MR: Phaser calculates the anisotropy so you can find the > value in the first lines of the log. > > > > > > Staraniso or the UCLA server are good to test if you have anisotropy. > Staraniso has a newer way of dealing with intensity falloff and accounting > for it. > > > > > > All the best > > > Vincent > > > > > > > > > > > > > > > On 13/10/2017 10:58, Paul Miller wrote: > > >> I had a similar problem to what you describe. In my case the dataset > was severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors > were stuck similar to yours but the map looked good. I was told by someone > with a much better appreciation of the theory than myself that the > anisotropy was causing the problem. > > >> > > >> It would be interesting to know from an expert in anisotropy e.g. the > creators of UCLA anisotropy server or Startaniso whether anisotropy can > cause this problem and whether there is any way around it. > > >> > > >> Cheers, Paul > > >> > > >> Paul Steven Mill
Re: [ccp4bb] High R/Rfree after MR
Dear Randy, On Fri, Oct 13, 2017 at 04:11:44PM +0100, Randy Read wrote: > Just to add to this point. The MR algorithms in Phaser are now able > to make better use of intensity data, which is particularly > important when you have any very weak data. Having weak data can’t > be avoided when you have serious anisotropy (or tNCS or a > combination of the two). Unfortunately, if you use amplitudes that > have been through the French & Wilson (truncate) algorithm, the real > variation in intensity is partially masked because the posterior > amplitude values are computed on the prior assumption that all the > reflections in a resolution shell have the same underlying intensity > distribution. Your "unfortunately" calsue may be the case with the UCLA server, but your statement is not true about what the STARANISO server (or STARANISO as invoked within autoPROC) does: as I already indicated in a reply to you a few months ago in this BB, the version of TRUNCATE in STARANISO applies the French & Wilson procedure with a prior Wilson probability whose expectation value for the intensity is modulated by the anisotropy of the dataset. This is clearly explained on the server - see http://staraniso.globalphasing.org/staraniso_about.html#step16 With best wishes, Gerard. -- > The UCLA server actually uses Phaser under the hood — what they add is to > turn the anisotropic B-values into suggested resolution limits in the > different directions. However, I don’t think they allow you yet to submit > intensities, which would be better. > > Best wishes, > > Randy Read > > - > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical ResearchTel: +44 1223 336500 > Wellcome Trust/MRC Building Fax: +44 1223 336827 > Hills RoadE-mail: > rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > > On 13 Oct 2017, at 10:29, vincent Chaptal wrote: > > > > Dear Gia and Paul, > > > > about anisotropy, one point to keep in mind is that it is not necessarily > > linked to the difference in resolution limits. > > In fact I am at the moment working on one of these cases, with extremely > > large difference in resolution limits, but relatively low anisotropy. > > Anisotropy is more a deviation from "normal" intensity falloff as a > > function of resolution. There is a thin difference/relationship between the > > two concepts that I think is worth investigating. > > > > we have performed a statistical analysis of this phenomenon and the paper > > is in revision at the moment, but if you want to know where your anisotropy > > stands in respect to all the other PDBs out there, feel free to contact me > > off list. > > You mention MR: Phaser calculates the anisotropy so you can find the value > > in the first lines of the log. > > > > Staraniso or the UCLA server are good to test if you have anisotropy. > > Staraniso has a newer way of dealing with intensity falloff and accounting > > for it. > > > > All the best > > Vincent > > > > > > > > > > On 13/10/2017 10:58, Paul Miller wrote: > >> I had a similar problem to what you describe. In my case the dataset was > >> severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were > >> stuck similar to yours but the map looked good. I was told by someone with > >> a much better appreciation of the theory than myself that the anisotropy > >> was causing the problem. > >> > >> It would be interesting to know from an expert in anisotropy e.g. the > >> creators of UCLA anisotropy server or Startaniso whether anisotropy can > >> cause this problem and whether there is any way around it. > >> > >> Cheers, Paul > >> > >> Paul Steven Miller (PhD) > >> Postdoctoral Researcher > >> University of Oxford > >> Wellcome Trust Centre for Human Genetics > >> Division of Structural Biology > >> Roosevelt Drive > >> Oxford > >> OX3 7BN > >> > >> > >> Original message > >> > >>> Date: Fri, 13 Oct 2017 10:30:22 +0200 > >>> From: CCP4 bulletin board > >>> (on behalf of Gianluca Cioci > >>> > >>> ) > >>> Subject: [ccp4bb] High R/Rfree after MR > >>> To: > >>> CCP4BB@JISCMAIL.AC.UK > >>> > >>> &g
Re: [ccp4bb] High R/Rfree after MR
Thank to all for the replies ! I have tried the UCLA server using the unmerged intensities from XDS and the verdict is strong anisotropy The Rfactors (after Phaser+Refmac) are only slightly lower for the Corrected. Non Corrected:Corrected R factor 0.3778 R factor 0.3732 R free 0.3871 R free 0.3816 I will make a few cycles of rebuilding and see if it makes any difference... Best regards, GIA Best regards, GIA On Fri, Oct 13, 2017 at 5:11 PM, Randy Read wrote: > Just to add to this point. The MR algorithms in Phaser are now able to > make better use of intensity data, which is particularly important when you > have any very weak data. Having weak data can’t be avoided when you have > serious anisotropy (or tNCS or a combination of the two). Unfortunately, > if you use amplitudes that have been through the French & Wilson (truncate) > algorithm, the real variation in intensity is partially masked because the > posterior amplitude values are computed on the prior assumption that all > the reflections in a resolution shell have the same underlying intensity > distribution. > > The UCLA server actually uses Phaser under the hood — what they add is to > turn the anisotropic B-values into suggested resolution limits in the > different directions. However, I don’t think they allow you yet to submit > intensities, which would be better. > > Best wishes, > > Randy Read > > - > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical ResearchTel: +44 1223 336500 > Wellcome Trust/MRC Building Fax: +44 1223 336827 > Hills Road > E-mail: rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > > On 13 Oct 2017, at 10:29, vincent Chaptal > wrote: > > > > Dear Gia and Paul, > > > > about anisotropy, one point to keep in mind is that it is not > necessarily linked to the difference in resolution limits. > > In fact I am at the moment working on one of these cases, with extremely > large difference in resolution limits, but relatively low anisotropy. > Anisotropy is more a deviation from "normal" intensity falloff as a > function of resolution. There is a thin difference/relationship between the > two concepts that I think is worth investigating. > > > > we have performed a statistical analysis of this phenomenon and the > paper is in revision at the moment, but if you want to know where your > anisotropy stands in respect to all the other PDBs out there, feel free to > contact me off list. > > You mention MR: Phaser calculates the anisotropy so you can find the > value in the first lines of the log. > > > > Staraniso or the UCLA server are good to test if you have anisotropy. > Staraniso has a newer way of dealing with intensity falloff and accounting > for it. > > > > All the best > > Vincent > > > > > > > > > > On 13/10/2017 10:58, Paul Miller wrote: > >> I had a similar problem to what you describe. In my case the dataset > was severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors > were stuck similar to yours but the map looked good. I was told by someone > with a much better appreciation of the theory than myself that the > anisotropy was causing the problem. > >> > >> It would be interesting to know from an expert in anisotropy e.g. the > creators of UCLA anisotropy server or Startaniso whether anisotropy can > cause this problem and whether there is any way around it. > >> > >> Cheers, Paul > >> > >> Paul Steven Miller (PhD) > >> Postdoctoral Researcher > >> University of Oxford > >> Wellcome Trust Centre for Human Genetics > >> Division of Structural Biology > >> Roosevelt Drive > >> Oxford > >> OX3 7BN > >> > >> > >> Original message > >> > >>> Date: Fri, 13 Oct 2017 10:30:22 +0200 > >>> From: CCP4 bulletin board > >>> (on behalf of Gianluca Cioci < > gianluca.ci...@gmail.com> > >>> ) > >>> Subject: [ccp4bb] High R/Rfree after MR > >>> To: > >>> CCP4BB@JISCMAIL.AC.UK > >>> > >>> > >>> Dear All, > >>> I am trying to refine a structure at 3.3A. Model has > >>> 60% identity to the target. Maps look OK (for 3.3A) > >>> and rebuilding in Coot is relatively > >>> straightforward. However, after some rebuilding > >>> cycles the R factors are stuck at 0.37/0.39 > >>> (REFMAC). > >>> XTRIAGE tells me that everything is normal (no twin, > >>> 98% completeness, R=3.5% in the low resolution bin), > >>> perhaps some anisotropy is present. > >>> I have already refined 2 homologous structures at > >>> resolutions going from 3.2 to 3.8 and there were no > >>> problems (final R ~ 0.21/0.24). > >>> Any advice ? > >>> Thanks, > >>> GIA > >>> > > > > -- > > Vincent Chaptal, PhD > > Institut de Biologie et Chimie des Protéines > > Drug Resistance and Membrane Proteins Laboratory > > 7 passage du Vercors > > 69007 LYON > > FRANCE > > +33 4 37 65 29 01 > > http://www.ibcp.fr > > > > >
Re: [ccp4bb] High R/Rfree after MR
Just to add to this point. The MR algorithms in Phaser are now able to make better use of intensity data, which is particularly important when you have any very weak data. Having weak data can’t be avoided when you have serious anisotropy (or tNCS or a combination of the two). Unfortunately, if you use amplitudes that have been through the French & Wilson (truncate) algorithm, the real variation in intensity is partially masked because the posterior amplitude values are computed on the prior assumption that all the reflections in a resolution shell have the same underlying intensity distribution. The UCLA server actually uses Phaser under the hood — what they add is to turn the anisotropic B-values into suggested resolution limits in the different directions. However, I don’t think they allow you yet to submit intensities, which would be better. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk > On 13 Oct 2017, at 10:29, vincent Chaptal wrote: > > Dear Gia and Paul, > > about anisotropy, one point to keep in mind is that it is not necessarily > linked to the difference in resolution limits. > In fact I am at the moment working on one of these cases, with extremely > large difference in resolution limits, but relatively low anisotropy. > Anisotropy is more a deviation from "normal" intensity falloff as a function > of resolution. There is a thin difference/relationship between the two > concepts that I think is worth investigating. > > we have performed a statistical analysis of this phenomenon and the paper is > in revision at the moment, but if you want to know where your anisotropy > stands in respect to all the other PDBs out there, feel free to contact me > off list. > You mention MR: Phaser calculates the anisotropy so you can find the value in > the first lines of the log. > > Staraniso or the UCLA server are good to test if you have anisotropy. > Staraniso has a newer way of dealing with intensity falloff and accounting > for it. > > All the best > Vincent > > > > > On 13/10/2017 10:58, Paul Miller wrote: >> I had a similar problem to what you describe. In my case the dataset was >> severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were >> stuck similar to yours but the map looked good. I was told by someone with a >> much better appreciation of the theory than myself that the anisotropy was >> causing the problem. >> >> It would be interesting to know from an expert in anisotropy e.g. the >> creators of UCLA anisotropy server or Startaniso whether anisotropy can >> cause this problem and whether there is any way around it. >> >> Cheers, Paul >> >> Paul Steven Miller (PhD) >> Postdoctoral Researcher >> University of Oxford >> Wellcome Trust Centre for Human Genetics >> Division of Structural Biology >> Roosevelt Drive >> Oxford >> OX3 7BN >> >> >> Original message >> >>> Date: Fri, 13 Oct 2017 10:30:22 +0200 >>> From: CCP4 bulletin board >>> (on behalf of Gianluca Cioci >>> >>> ) >>> Subject: [ccp4bb] High R/Rfree after MR >>> To: >>> CCP4BB@JISCMAIL.AC.UK >>> >>> >>> Dear All, >>> I am trying to refine a structure at 3.3A. Model has >>> 60% identity to the target. Maps look OK (for 3.3A) >>> and rebuilding in Coot is relatively >>> straightforward. However, after some rebuilding >>> cycles the R factors are stuck at 0.37/0.39 >>> (REFMAC). >>> XTRIAGE tells me that everything is normal (no twin, >>> 98% completeness, R=3.5% in the low resolution bin), >>> perhaps some anisotropy is present. >>> I have already refined 2 homologous structures at >>> resolutions going from 3.2 to 3.8 and there were no >>> problems (final R ~ 0.21/0.24). >>> Any advice ? >>> Thanks, >>> GIA >>> > > -- > Vincent Chaptal, PhD > Institut de Biologie et Chimie des Protéines > Drug Resistance and Membrane Proteins Laboratory > 7 passage du Vercors > 69007 LYON > FRANCE > +33 4 37 65 29 01 > http://www.ibcp.fr > >
[ccp4bb] AW: [ccp4bb] High R/Rfree after MR
Dear GIA, In addition to the anisotropy, I would also check your diffraction images and make sure that there are no (even hardly perceptible) ice rings present. Depending on how the data processing software handled this, they may cause high Rfactors in the range you mentioned. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Gianluca Cioci Gesendet: Freitag, 13. Oktober 2017 10:30 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] High R/Rfree after MR Dear All, I am trying to refine a structure at 3.3A. Model has 60% identity to the target. Maps look OK (for 3.3A) and rebuilding in Coot is relatively straightforward. However, after some rebuilding cycles the R factors are stuck at 0.37/0.39 (REFMAC). XTRIAGE tells me that everything is normal (no twin, 98% completeness, R=3.5% in the low resolution bin), perhaps some anisotropy is present. I have already refined 2 homologous structures at resolutions going from 3.2 to 3.8 and there were no problems (final R ~ 0.21/0.24). Any advice ? Thanks, GIA
Re: [ccp4bb] High R/Rfree after MR
Dear Gia and Paul, about anisotropy, one point to keep in mind is that it is not necessarily linked to the difference in resolution limits. In fact I am at the moment working on one of these cases, with extremely large difference in resolution limits, but relatively low anisotropy. Anisotropy is more a deviation from "normal" intensity falloff as a function of resolution. There is a thin difference/relationship between the two concepts that I think is worth investigating. we have performed a statistical analysis of this phenomenon and the paper is in revision at the moment, but if you want to know where your anisotropy stands in respect to all the other PDBs out there, feel free to contact me off list. You mention MR: Phaser calculates the anisotropy so you can find the value in the first lines of the log. Staraniso or the UCLA server are good to test if you have anisotropy. Staraniso has a newer way of dealing with intensity falloff and accounting for it. All the best Vincent On 13/10/2017 10:58, Paul Miller wrote: I had a similar problem to what you describe. In my case the dataset was severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck similar to yours but the map looked good. I was told by someone with a much better appreciation of the theory than myself that the anisotropy was causing the problem. It would be interesting to know from an expert in anisotropy e.g. the creators of UCLA anisotropy server or Startaniso whether anisotropy can cause this problem and whether there is any way around it. Cheers, Paul Paul Steven Miller (PhD) Postdoctoral Researcher University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN Original message Date: Fri, 13 Oct 2017 10:30:22 +0200 From: CCP4 bulletin board (on behalf of Gianluca Cioci ) Subject: [ccp4bb] High R/Rfree after MR To: CCP4BB@JISCMAIL.AC.UK Dear All, I am trying to refine a structure at 3.3A. Model has 60% identity to the target. Maps look OK (for 3.3A) and rebuilding in Coot is relatively straightforward. However, after some rebuilding cycles the R factors are stuck at 0.37/0.39 (REFMAC). XTRIAGE tells me that everything is normal (no twin, 98% completeness, R=3.5% in the low resolution bin), perhaps some anisotropy is present. I have already refined 2 homologous structures at resolutions going from 3.2 to 3.8 and there were no problems (final R ~ 0.21/0.24). Any advice ? Thanks, GIA -- Vincent Chaptal, PhD Institut de Biologie et Chimie des Protéines Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://www.ibcp.fr
Re: [ccp4bb] High R/Rfree after MR
Gianluca, you could become an expert yourself, by trying - the UCLA anisotropy server - STARANISO with these data, and report here what the outcome in each case is - Rwork/Rfree, map appearance, ... My personal experience is that both refmac and phenix.refine deal well with moderate anisotropy (but there is room for improvement, e.g. current phenix.refine does not take the sigmas into account). In cases of severe anisotropy I had good results with STARANISO - better phasing, better maps, better refinement. best, Kay On Fri, 13 Oct 2017 09:58:39 +0100, Paul Miller wrote: >I had a similar problem to what you describe. In my case the dataset was >severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck >similar to yours but the map looked good. I was told by someone with a much >better appreciation of the theory than myself that the anisotropy was causing >the problem. > >It would be interesting to know from an expert in anisotropy e.g. the creators >of UCLA anisotropy server or Startaniso whether anisotropy can cause this >problem and whether there is any way around it. > >Cheers, Paul > >Paul Steven Miller (PhD) >Postdoctoral Researcher >University of Oxford >Wellcome Trust Centre for Human Genetics >Division of Structural Biology >Roosevelt Drive >Oxford >OX3 7BN > > > Original message >>Date: Fri, 13 Oct 2017 10:30:22 +0200 >>From: CCP4 bulletin board (on behalf of Gianluca >>Cioci ) >>Subject: [ccp4bb] High R/Rfree after MR >>To: CCP4BB@JISCMAIL.AC.UK >> >> Dear All, >> I am trying to refine a structure at 3.3A. Model has >> 60% identity to the target. Maps look OK (for 3.3A) >> and rebuilding in Coot is relatively >> straightforward. However, after some rebuilding >> cycles the R factors are stuck at 0.37/0.39 >> (REFMAC).� >> XTRIAGE tells me that everything is normal (no twin, >> 98% completeness, R=3.5% in the low resolution bin), >> perhaps some anisotropy is present.� >> I have already refined 2 homologous structures at >> resolutions going from 3.2 to 3.8 and there were no >> problems (final R ~ 0.21/0.24).� >> Any advice ? >> Thanks, >> GIA
Re: [ccp4bb] High R/Rfree after MR
I had a similar problem to what you describe. In my case the dataset was severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck similar to yours but the map looked good. I was told by someone with a much better appreciation of the theory than myself that the anisotropy was causing the problem. It would be interesting to know from an expert in anisotropy e.g. the creators of UCLA anisotropy server or Startaniso whether anisotropy can cause this problem and whether there is any way around it. Cheers, Paul Paul Steven Miller (PhD) Postdoctoral Researcher University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN Original message >Date: Fri, 13 Oct 2017 10:30:22 +0200 >From: CCP4 bulletin board (on behalf of Gianluca Cioci >) >Subject: [ccp4bb] High R/Rfree after MR >To: CCP4BB@JISCMAIL.AC.UK > > Dear All, > I am trying to refine a structure at 3.3A. Model has > 60% identity to the target. Maps look OK (for 3.3A) > and rebuilding in Coot is relatively > straightforward. However, after some rebuilding > cycles the R factors are stuck at 0.37/0.39 > (REFMAC). > XTRIAGE tells me that everything is normal (no twin, > 98% completeness, R=3.5% in the low resolution bin), > perhaps some anisotropy is present. > I have already refined 2 homologous structures at > resolutions going from 3.2 to 3.8 and there were no > problems (final R ~ 0.21/0.24). > Any advice ? > Thanks, > GIA
[ccp4bb] High R/Rfree after MR
Dear All, I am trying to refine a structure at 3.3A. Model has 60% identity to the target. Maps look OK (for 3.3A) and rebuilding in Coot is relatively straightforward. However, after some rebuilding cycles the R factors are stuck at 0.37/0.39 (REFMAC). XTRIAGE tells me that everything is normal (no twin, 98% completeness, R=3.5% in the low resolution bin), perhaps some anisotropy is present. I have already refined 2 homologous structures at resolutions going from 3.2 to 3.8 and there were no problems (final R ~ 0.21/0.24). Any advice ? Thanks, GIA