Hi all, I have a structure refined to 2.5 A. After simulated annealing (start temp = 5000, cool rate = 25, final temp = 100, include TLS refinement and NCS restraint), my R and Rfree are 24.6% and 28.7% respectively. I then turned off the SA in the generated def file and carried on the combined refinement. My R and Rfree went up to 25% and 29.2% respectively. Why did the Rfree go up after refinement? In the remarks of the output pdb file, I have one line, "REMARK twinned : True". What does this mean? The RMSD of bonds and angles after either SA or combined refinement are about 0.006 and 0.851 respectively. Don't you guys think the geometry restraints are too tight? How do I change the weight in PHENIX? Thanks. Jianghai +++++++++++++++++++++++++++++++ Jianghai Zhu, Ph.D CBR Institute for Biomedical Research Department of Pathology Harvard Medical School 200 Longwood Ave., Boston, MA 02115 Ph: 617-278-3211 Fx: 618-278-3030 +++++++++++++++++++++++++++++++
In the remarks of the output pdb file, I have one line, "REMARK twinned : True". What does this mean?
phenix.refine does a basic check to see whether or not the data is twinned. In this case, the refinement engine thinks data data might be twinned. Please run mmtbx.xtriage refinemenet_results_data.mtz obs=FOBS calc=FMODEL to get more clues why phenix.refine thinks why the data might be twinned. If you have problems intepreting the xtriage output, let me know, I'll give you a hand. HTH Peter
Hi Jianghai Zhu, thanks for your questions!
<>I have a structure refined to 2.5 A. After simulated annealing (start temp = 5000, cool rate = 25, final temp = 100, include TLS refinement and NCS restraint), my R and Rfree are 24.6% and 28.7% respectively. I then turned off the SA in the generated def file and carried on the combined refinement. My R and Rfree went up to 25% and 29.2% respectively. Why did the Rfree go up after refinement?
The difference between 28.7% and 29.2% is not large (=0.5%). If you run a set of identical refinement jobs with different random seed you will get the distribution of final R-factors ranging within 0.5%. So I would not worry too much about this. However, I would try different refinement strategy, for example: 1) you do SA refinement first; 2) then you do regular refinement + TLS.
The RMSD of bonds and angles after either SA or combined refinement are about 0.006 and 0.851 respectively. Don't you guys think the geometry restraints are too tight?
At 2.5A resolution I wouldn't worry about these values. The resolution is low enough to keep the geometry tight.
How do I change the weight in PHENIX?
However, if you want you can play with the target weights. The total target function in phenix.refine for the coordinates refinement is : T = wxc_scale * wxc * Exray + wc * Egeom, where wxc is determined automatically and wxc_scale is a parameter that the user can modify. So, if you want to make the X-ray term stronger, then obviously you need to increase the wxc_scale value. By default it is 0.5 (see parameters file under the scope refinement.target_weights). Try several wxc_scale and pick the refinement outcome you like best. Let us know if you have more problems / questions. Cheers, Pavel.
Thanks.
However, I would try different refinement strategy, for example: 1) you do SA refinement first; 2) then you do regular refinement + TLS.
How to do SA refinement only?
The RMSD of bonds and angles after either SA or combined refinement are about 0.006 and 0.851 respectively. Don't you guys think the geometry restraints are too tight?
At 2.5A resolution I wouldn't worry about these values. The resolution is low enough to keep the geometry tight.
I thought the goal of the refinement for geometry is 0.02 and 2 for rmsd bonds and angle. Another questions. After SA or refinement, the B factors vary a lot. In the same residue, B factors jump from 5 to 70. and for the next residue, B factors are from 40 to 90. The B factors don't look like well restrained. Are these numbers normal? I don't see this kind behavior of B factor in other refinement programs. Jianghai
Hi Jianghai Zhu,
However, I would try different refinement strategy, for example: 1) you do SA refinement first; 2) then you do regular refinement + TLS.
How to do SA refinement only?
To run the default protocol + SA: % phenix.refine data.hkl model.pdb simulated_annealing=true For more information see phenix.refine manual: http://phenix-online.org/download/documentation/phenix/phenix/structure_refi...
The RMSD of bonds and angles after either SA or combined refinement are about 0.006 and 0.851 respectively. Don't you guys think the geometry restraints are too tight?
At 2.5A resolution I wouldn't worry about these values. The resolution is low enough to keep the geometry tight.
I thought the goal of the refinement for geometry is 0.02 and 2 for rmsd bonds and angle.
This is resolution dependent. At low resolution your stereochemistry rmsd should be close to ideal. At high resolution it makes sense to have the geometry more relaxed. Anyway, this should be justified by the amount of data and reasonable values of Rfree. The automatic weight calculation in phneix.refine provides the optimal weight in most of the cases. If you like to change the default weight, you can do so as I mentioned in previous email. This is described in the Manual as well.
Another questions. After SA or refinement, the B factors vary a lot. In the same residue, B factors jump from 5 to 70. and for the next residue, B factors are from 40 to 90. The B factors don't look like well restrained. Are these numbers normal? I don't see this kind behavior of B factor in other refinement programs.
In this case you may want to increase ADP restraints contribution by decreasing X-ray term weight. To do so, decrease wxu_scale value. By default wxu_scale=1.0. Try smaller values and see your B-factors. See Manual for more details. Thanks for your questions ! Pavel.
participants (3)
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Jianghai Zhu -
Pavel Afonine -
Peter Zwart