[ccp4bb] AW: [ccp4bb] Stop Refmac from refining B factors?
Dear Eva and Harry and others, I am not sure that an overall B-factor is the best solution for a 3.2 Å structure. In general, the true B-factors will vary a lot for different parts of the protein and poor diffracting proteins often have parts which are partially or completely disordered. An overall B-factor is a very poor estimate of such a true B-factor distribution and together with the low-resolution data will create a lot of model bias i.e. very nice density for side chains which are, in fact, disordered. I have struggled a long time with a 3.2 Å antithrombin data set and as luck would have it, the most interesting and unknown part of the structure was a loop with extremely high temperature factors. All standard protocols I tried produced very nice density for the model I put in, but did not tell me anything of how to build the unknown parts. A side remark by Randy Read finally helped me to solve this problem, which was to reduce model bias by using very tight geometric restraints (e.g. not allow the xyz positions produce bias) and individual temperature factors. What I oberved was that wrongly fitted residues would be pushed out of the density due to the tight geometric restraints, the artificially good electron density of some flexible parts would get weaker and most importantly: electron density appeared were I could fit the important reactive site loop. It worked in this case, it may not work in other cases and at 3.2 Å one should always proceed very cautiously and try to use whatever information (biochemical, mutations) is available to validate the model. This was my experience and I have never used an overall B-factor afterwards. Herman Schreuder Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Harry M. Greenblatt Gesendet: Mittwoch, 18. April 2007 14:38 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Stop Refmac from refining B factors? BSD Dear Eva, You should be refining an overall temperature factor at that resolution. It's one of the choices in the list, instead of isotropic. Hi, I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%. Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac flattens them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution. Here is what I tried further: * I de-selected Refine isotropic temperature factors in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?) * I played around with the geometric parameters. If I select the B-factor values there (the keywords are TEMP|BFAC wbskalsigb1sigb2sigb3sigb4), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even flatter). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs. Thanks for any thoughts on this, Eva - Harry M. Greenblatt Staff Scientist Dept of Structural Biology [EMAIL PROTECTED] Weizmann Institute of SciencePhone: 972-8-934-3625 Rehovot, 76100 Facsimile: 972-8-934-4159 Israel
Re: [ccp4bb] Stop Refmac from refining B factors?
On 18 Apr 2007, at 14:39, Eva Kirchner wrote: (But I'm still curious about the B-factor refinement when there is no REFI BREF ISOT in the com-file...) Eva, Refmac internal default is REFI BREF ISOT that's why even if you remove the above line from the com file (or deselect that option from the interface) it still does ISOT BREF refinement. It does tell you that though if you look at the log file there's a bit that says ... Method of minimisation : Sparse Matrix Experimental sigmas used for weighting Number of Bins and width:20 0.0080 Refinement of individual isotropic Bfactors .. what do you have there when you deselect the B fact option from the interface? I agree with Herman that at 3.2 A isotropic B values refinement can be useful. Roberto Eva 2007/4/18, Mischa Machius [EMAIL PROTECTED]: Like Harry said, it is not justified to do individual B factor refinement at that resolution. Well, you can do it, but you'll end up with funny results, such as what are observing right now. Still, from a pragmatic point of view, individual B factor refinement in cases like these can have a positive effect on the electron density. However, keep in mind that the resulting B factors may physically not be very meaningful. In the end, you'll have to switch to grouped B factor refinement, or you risk nasty comments from an attentive mentor or reviewer (and rightly so). Hope that helps. Best - MM On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote: Hi, I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%. Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac flattens them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution. Here is what I tried further: * I de-selected Refine isotropic temperature factors in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?) * I played around with the geometric parameters. If I select the B- factor values there (the keywords are TEMP|BFAC wbskalsigb1sigb2sigb3sigb4), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even flatter). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs. Thanks for any thoughts on this, Eva -- -- Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Stop Refmac from refining B factors?
Harry M. Greenblatt wrote: You should be refining an overall temperature factor at that resolution. It's one of the choices in the list, instead of isotropic. I disagree with this. At that (3.2 Angstrom) resolution I've often found than a tightly restrained individual B-factor refinement gives a significantly lower R-free than a single overall B-factor. I also prefer it to grouped B-factors in CNS, because the latter are not geometrically restrained and show a lot of physically unreasonable waywardness (although often, similar R-free as B-individual). Individual B's can also be restrained by non-crystallographic symmetry and as far as I can tell grouped B's are not. I think one has to explore all possibilities rather than take one fixed approach to working at modest resolutions, and the optimal solution is likely to be different for different structures. Phil Jeffrey Princeton, NJ Hi, I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%. Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac flattens them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution. Here is what I tried further: * I de-selected Refine isotropic temperature factors in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?) * I played around with the geometric parameters. If I select the B-factor values there (the keywords are TEMP|BFAC wbskalsigb1sigb2sigb3sigb4), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even flatter). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs. Thanks for any thoughts on this, Eva - Harry M. Greenblatt Staff Scientist Dept of Structural Biology [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] Weizmann Institute of SciencePhone: 972-8-934-3625 Rehovot, 76100 Facsimile: 972-8-934-4159 Israel
Re: [ccp4bb] Stop Refmac from refining B factors?
Thank you Roberto, I saw that line in the log file, too. So it is as I feared: Refmac cannot be stopped from refining B-factors ;-) Maybe I'll use CNS... Eva 2007/4/18, Roberto Steiner [EMAIL PROTECTED]: On 18 Apr 2007, at 14:39, Eva Kirchner wrote: (But I'm still curious about the B-factor refinement when there is no REFI BREF ISOT in the com-file...) Eva, Refmac internal default is REFI BREF ISOT that's why even if you remove the above line from the com file (or deselect that option from the interface) it still does ISOT BREF refinement. It does tell you that though if you look at the log file there's a bit that says ... Method of minimisation : Sparse Matrix Experimental sigmas used for weighting Number of Bins and width:20 0.0080 Refinement of individual isotropic Bfactors .. what do you have there when you deselect the B fact option from the interface? I agree with Herman that at 3.2 A isotropic B values refinement can be useful. Roberto Eva 2007/4/18, Mischa Machius [EMAIL PROTECTED]: Like Harry said, it is not justified to do individual B factor refinement at that resolution. Well, you can do it, but you'll end up with funny results, such as what are observing right now. Still, from a pragmatic point of view, individual B factor refinement in cases like these can have a positive effect on the electron density. However, keep in mind that the resulting B factors may physically not be very meaningful. In the end, you'll have to switch to grouped B factor refinement, or you risk nasty comments from an attentive mentor or reviewer (and rightly so). Hope that helps. Best - MM On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote: Hi, I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019 , a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%. Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac flattens them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution. Here is what I tried further: * I de-selected Refine isotropic temperature factors in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?) * I played around with the geometric parameters. If I select the B-factor values there (the keywords are TEMP|BFAC wbskalsigb1sigb2sigb3sigb4), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even flatter). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs. Thanks for any thoughts on this, Eva Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Stop Refmac from refining B factors?
On Wednesday 18 April 2007 07:40, Phil Jeffrey wrote: Harry M. Greenblatt wrote: You should be refining an overall temperature factor at that resolution. It's one of the choices in the list, instead of isotropic. I disagree with this. At that (3.2 Angstrom) resolution I've often found than a tightly restrained individual B-factor refinement gives a significantly lower R-free than a single overall B-factor. I also prefer it to grouped B-factors in CNS My suggestion (as you can probably guess): Step 1 Set the geometric restraints very tight, but the thermal restraints not so tight. Refine individual or group B's (I doubt it matters which). Step 2 Take that model, unrealistic as it is, and feed it to the TLSMD server for automated analysis of the distribution of B factors in real space. Tell the server to use the backbone B's only (it's a tick-box on the 'advanced options' page). The output should suggest to you which portions of your structure are suitable for treatment as coherent groups; some will be relatively well-ordered, others may be waving around wildly. Step 3 Go back to refmac and perform a multi-group TLS refinement using the groups suggested by TLSMD. Because this is low resolution, you should refine a pure TLS model. That is, reset all the individual B's to some constant value and let the TLS model alone describe their variation. Success is never guaranteed, but in test cases we have found that for low resolution structures a multi-group pure TLS model can give better R values than individual isotropic B refinement, while ensuring that the distribution of B's adheres to a physically plausible model. Ethan Merritt , because the latter are not geometrically restrained and show a lot of physically unreasonable waywardness (although often, similar R-free as B-individual). Individual B's can also be restrained by non-crystallographic symmetry and as far as I can tell grouped B's are not. I think one has to explore all possibilities rather than take one fixed approach to working at modest resolutions, and the optimal solution is likely to be different for different structures. Phil Jeffrey Princeton, NJ Hi, I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%. Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac flattens them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution. Here is what I tried further: * I de-selected Refine isotropic temperature factors in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?) * I played around with the geometric parameters. If I select the B-factor values there (the keywords are TEMP|BFAC wbskalsigb1sigb2sigb3sigb4), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even flatter). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs. Thanks for any thoughts on this, Eva -- Ethan A MerrittCourier Deliveries: 1959 NE Pacific Dept of Biochemistry Health Sciences Building University of Washington - Seattle WA 98195-7742
