Gap between R and Rfree

Young-Jin Cho

25 Feb 2010 25 Feb '10

5:17 p.m.

Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful. Thanks in advance, Young-Jin

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Phil Jeffrey

25 Feb 25 Feb

5:33 p.m.

More information would be very helpful for giving detailed suggestions, however: 0. Use Molprobity or the Validation tools in Coot to look at your structure and see what errors you can fix. 1. What is your geometry like ? Try reducing wxc_scale (try wxc_scale=0.5) and see what happens. 2. What is the variation of B-factors across bonds ? Try reducing wxu_scale - perhaps wxu_scale=0.5 or less. 2a. phenix.refine is autocratic about wxu_scale if you're using TLS so setting it yourself does not help. 3. Since R-free of 0.298 is a little high for a 2.5 Angstrom structure, you should carefully check your model. Perhaps you have a high proportion of disordered structure, which can cause this. If you feel that your model is correct, you could explore using TLS on your model. Potential TLS groups can be found using the TLSMD server at: http://skuld.bmsc.washington.edu/~tlsmd/ although the results should always be filtered through a common-sense definition of domain/group boundaries (e.g. don't break in the middle of helices unless there's a good reason to). Phil Jeffrey Princeton Young-Jin Cho wrote:

...

Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

Young-Jin Cho

5:41 p.m.

Thanks for your suggestions, Phill. When I did Ramachandran analysis, it shows 96.60% in preferred region and 2.62% in allowed regions, and 0.79% is in outlier. Geometry looks Ok. I also did optimize_wxc and optimize_wxu, and tls option. it didn't help much. Now I am trying to scale down. Thanks again. Young-Jin 0. Use Molprobity or the Validation tools in Coot to look at your structure and see what errors you can fix. 1. What is your geometry like ? Try reducing wxc_scale (try wxc_scale=0.5) and see what happens. 2. What is the variation of B-factors across bonds ? Try reducing wxu_scale - perhaps wxu_scale=0.5 or less. 2a. phenix.refine is autocratic about wxu_scale if you're using TLS so setting it yourself does not help. 3. Since R-free of 0.298 is a little high for a 2.5 Angstrom structure, you should carefully check your model. Perhaps you have a high proportion of disordered structure, which can cause this. If you feel that your model is correct, you could explore using TLS on your model. Potential TLS groups can be found using the TLSMD server at: http://skuld.bmsc.washington.edu/~tlsmd/ although the results should always be filtered through a common-sense definition of domain/group boundaries (e.g. don't break in the middle of helices unless there's a good reason to). Phil Jeffrey Princeton Young-Jin Cho wrote:

...

Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb -- Postdoctoral Fellow, Kern Lab Biochemistry Department/HHMI Brandeis University

Kendall Nettles

6:35 p.m.

Did you check your data Kendall On Feb 25, 2010, at 12:43 PM, "Young-Jin Cho" wrote:

...

Thanks for your suggestions, Phill. When I did Ramachandran analysis, it shows 96.60% in preferred region and 2.62% in allowed regions, and 0.79% is in outlier. Geometry looks Ok. I also did optimize_wxc and optimize_wxu, and tls option. it didn't help much. Now I am trying to scale down.

Thanks again.

Young-Jin

0. Use Molprobity or the Validation tools in Coot to look at your structure and see what errors you can fix. 1. What is your geometry like ? Try reducing wxc_scale (try wxc_scale=0.5) and see what happens. 2. What is the variation of B-factors across bonds ? Try reducing wxu_scale - perhaps wxu_scale=0.5 or less. 2a. phenix.refine is autocratic about wxu_scale if you're using TLS so setting it yourself does not help. 3. Since R-free of 0.298 is a little high for a 2.5 Angstrom structure, you should carefully check your model. Perhaps you have a high proportion of disordered structure, which can cause this. If you feel that your model is correct, you could explore using TLS on your model. Potential TLS groups can be found using the TLSMD server at: http://skuld.bmsc.washington.edu/~tlsmd/ although the results should always be filtered through a common-sense definition of domain/group boundaries (e.g. don't break in the middle of helices unless there's a good reason to).

Phil Jeffrey Princeton

Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

-- Postdoctoral Fellow, Kern Lab Biochemistry Department/HHMI Brandeis University _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Young-Jin Cho

7:45 p.m.

Thanks everyone. First, I checked my data and showed almost no anisotropy. Second, I tried both refmac and phenix, had the similar results. Third, ncs may be the key... I re-processed hkl and cut the resolution and did ncs=true. It first generated error message and suggested to put distance limit upto 3. Then now the gap between R and Rfree is reasonable (.2834 and .3361 without substrate and water). However I tried my previous pdb that has everything with old mtz file, Final R-work = 0.2389, R-free = 0.3158. In this case, I had to expand my limit upto 6. I still need to look more or do further refine from newly generated mtz file though, (sorry that's why I was late for responding.... I wanted to confirm it). Anyway, seemingly setting up 'ncs' as Pavel mentioned is most possible answer to my case. Thanks again. YoungJin ----- Original Message ----- From: "Kendall Nettles" To: "PHENIX user mailing list" Sent: Thursday, February 25, 2010 1:35:10 PM GMT -05:00 US/Canada Eastern Subject: Re: [phenixbb] Gap between R and Rfree Did you check your data Kendall On Feb 25, 2010, at 12:43 PM, "Young-Jin Cho" wrote:

...

Thanks for your suggestions, Phill. When I did Ramachandran analysis, it shows 96.60% in preferred region and 2.62% in allowed regions, and 0.79% is in outlier. Geometry looks Ok. I also did optimize_wxc and optimize_wxu, and tls option. it didn't help much. Now I am trying to scale down.

Thanks again.

Young-Jin

0. Use Molprobity or the Validation tools in Coot to look at your structure and see what errors you can fix. 1. What is your geometry like ? Try reducing wxc_scale (try wxc_scale=0.5) and see what happens. 2. What is the variation of B-factors across bonds ? Try reducing wxu_scale - perhaps wxu_scale=0.5 or less. 2a. phenix.refine is autocratic about wxu_scale if you're using TLS so setting it yourself does not help. 3. Since R-free of 0.298 is a little high for a 2.5 Angstrom structure, you should carefully check your model. Perhaps you have a high proportion of disordered structure, which can cause this. If you feel that your model is correct, you could explore using TLS on your model. Potential TLS groups can be found using the TLSMD server at: http://skuld.bmsc.washington.edu/~tlsmd/ although the results should always be filtered through a common-sense definition of domain/group boundaries (e.g. don't break in the middle of helices unless there's a good reason to).

Phil Jeffrey Princeton

Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

Pavel Afonine

7:58 p.m.

Hi Young-Jin,

...

Third, ncs may be the key... I re-processed hkl and cut the resolution

I'm wondering why you cut the resolution?

...

and did ncs=true. It first generated error message and suggested to put distance limit upto 3.

With ncs=true phenix.refine will determine the NCS groups automatically. Since the currently used algorithm is very simple the NCS groups may not be chosen optimally (making it smart is in to-do list). This is why it is a good idea to check the automatically determined NCS groups, especially when you see a message that asks you to push the distance limit to a larger value. You can look in .geo file (the file that lists all the geometry restraints used in refinement) for something like EXCESSIVE DISTANCE to see affected atoms and then check the map to see if these atoms should be forced to obey NCS and not represent the individuality of NCS related groups and therefore need to be excluded from NCS restraints. Good luck! Pavel.

Nathaniel Echols

8:05 p.m.

On Feb 25, 2010, at 11:45 AM, Young-Jin Cho wrote:

...

Thanks everyone. First, I checked my data and showed almost no anisotropy. Second, I tried both refmac and phenix, had the similar results. Third, ncs may be the key... I re-processed hkl and cut the resolution and did ncs=true. It first generated error message and suggested to put distance limit upto 3. Then now the gap between R and Rfree is reasonable (.2834 and .3361 without substrate and water). However I tried my previous pdb that has everything with old mtz file, Final R-work = 0.2389, R-free = 0.3158. In this case, I had to expand my limit upto 6.

I still need to look more or do further refine from newly generated mtz file though, (sorry that's why I was late for responding.... I wanted to confirm it). Anyway, seemingly setting up 'ncs' as Pavel mentioned is most possible answer to my case.

Other less likely (but still possible) things to look for: 1. Twinning with a small twin fraction - this can be easy to miss if your maps look decent and the R-free isn't ridiculously high. 2. Undiagnosed translational pseudosymmetry. I think I spent several months of grad school trying to refine a structure like this because I didn't realize what the extra spots in my images meant. (In my defense, they were more smudges than "spots", but R-free dropped a few percent lower when I reprocessed and re-refined with the correct symmetry.) General question: this topic comes up over and over again here and on ccp4bb. Is there a general FAQ list anywhere that covers all of these issues? (I will add this to the Phenix FAQ, along with the answer to "how do I tell if my R-free is good", but most of the same answers apply to any software, not just Phenix.) Nat ------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]

Pavel Afonine

8:20 p.m.

...

General question: this topic comes up over and over again here and on ccp4bb. Is there a general FAQ list anywhere that covers all of these issues? (I will add this to the Phenix FAQ, along with the answer to "how do I tell if my R-free is good", but most of the same answers apply to any software, not just Phenix.)

The question "how do I tell if my R-free is good" that keeps coming up from time to time motivated us to write this paper: Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009). The corresponding tool, called POLYGON, is available in PHENIX. Related utility, "PDB Statistics Overview" is available in PHENIX as well. So at this point we can automatically tell if the R-factors are good or not, and the next step in automation would be to take right refinement decisions based on this. Work in progress -:) Pavel.

Kendall Nettles

6:37 p.m.

Did you check your data for anisotropy at the UCLA server? This solved a similar problem for me Kendall On Feb 25, 2010, at 12:43 PM, "Young-Jin Cho" wrote:

...

Thanks for your suggestions, Phill. When I did Ramachandran analysis, it shows 96.60% in preferred region and 2.62% in allowed regions, and 0.79% is in outlier. Geometry looks Ok. I also did optimize_wxc and optimize_wxu, and tls option. it didn't help much. Now I am trying to scale down.

Thanks again.

Young-Jin

0. Use Molprobity or the Validation tools in Coot to look at your structure and see what errors you can fix. 1. What is your geometry like ? Try reducing wxc_scale (try wxc_scale=0.5) and see what happens. 2. What is the variation of B-factors across bonds ? Try reducing wxu_scale - perhaps wxu_scale=0.5 or less. 2a. phenix.refine is autocratic about wxu_scale if you're using TLS so setting it yourself does not help. 3. Since R-free of 0.298 is a little high for a 2.5 Angstrom structure, you should carefully check your model. Perhaps you have a high proportion of disordered structure, which can cause this. If you feel that your model is correct, you could explore using TLS on your model. Potential TLS groups can be found using the TLSMD server at: http://skuld.bmsc.washington.edu/~tlsmd/ although the results should always be filtered through a common-sense definition of domain/group boundaries (e.g. don't break in the middle of helices unless there's a good reason to).

Phil Jeffrey Princeton

Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

-- Postdoctoral Fellow, Kern Lab Biochemistry Department/HHMI Brandeis University _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Maia Cherney

6:38 p.m.

My suggestion is try a different refinement program. Maia Phil Jeffrey wrote:

...

More information would be very helpful for giving detailed suggestions, however:

0. Use Molprobity or the Validation tools in Coot to look at your structure and see what errors you can fix. 1. What is your geometry like ? Try reducing wxc_scale (try wxc_scale=0.5) and see what happens. 2. What is the variation of B-factors across bonds ? Try reducing wxu_scale - perhaps wxu_scale=0.5 or less. 2a. phenix.refine is autocratic about wxu_scale if you're using TLS so setting it yourself does not help. 3. Since R-free of 0.298 is a little high for a 2.5 Angstrom structure, you should carefully check your model. Perhaps you have a high proportion of disordered structure, which can cause this. If you feel that your model is correct, you could explore using TLS on your model. Potential TLS groups can be found using the TLSMD server at: http://skuld.bmsc.washington.edu/~tlsmd/ although the results should always be filtered through a common-sense definition of domain/group boundaries (e.g. don't break in the middle of helices unless there's a good reason to).

Phil Jeffrey Princeton

Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

9:42 p.m.

Don't forget then to use the correct work/test reflection flags. Some programs by default use 0 as test and 1 as work, some use 1 as test and 0 as work, +1 and -1 can be as well, some generate binary flags: for example 0 and 1 or 1 and -1, and some create an array of values from 0 to 19. Finally, some programs detect the above cases automatically, but under certain and rare circumstances the automatic choice may be wrong. Not to mention the TLS (if used). Pavel. On 2/25/10 10:38 AM, Maia Cherney wrote:

...

My suggestion is try a different refinement program.

Maia

Pavel Afonine

5:43 p.m.

Hi Young-Jin, the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following: Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29 which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting. A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version. Let me now if you have any questions. Pavel. On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...

Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Francis E Reyes

5:45 p.m.

Pavel What phenix utility produces such information? Thx FR On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...

Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

7:39 p.m.

Hi Francis, in PHENIX GUI: "Validation" -> "PDB Statistics Overview" or: in PHENIX GUI: "Validation" -> "POLYGON" The underlying idea is published here: Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009) I can add a command line version if anyone is interested. Pavel. On 2/25/10 9:45 AM, Francis E Reyes wrote:

...

Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Simon Kolstoe

10:01 p.m.

oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI? Simon On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...

Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Nathaniel Echols

10:50 p.m.

On Feb 25, 2010, at 2:01 PM, Simon Kolstoe wrote:

...

oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Not sure if you found this already, but the GUI can save the image as PNG, which can be converted into pretty much anything. Doing this automatically from the command line is certainly possible - the code is relatively modular and was written with this in mind - but it's actually a fair amount of work, because the image is ultimately drawn directly by wxPython (i.e. it isn't simply an image embedded in the window). (Plain text output, on the other hand, is very simple.) I would actually prefer to switch entirely to a scalable, device-independent format such as PS or PDF (which the GUI could then display), but it will take a while for this to migrate up my to-do list. The simple text output like the histograms Pavel shows are much easier to do, probably just a matter of pulling together code fragments. I'm pretty sure I could produce graphical output for these entirely on the command line (see the PDB Statistics GUI for an example of what they'd look like), since the drawing of these is handled by a separate module. But this isn't going to save much time over feeding the raw numbers into Excel and making your own graphs. Nat ------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]

Pavel Afonine

26 Feb 26 Feb

1:49 a.m.

Hi Simon, the next available PHENIX nightly build will have this command: phenix.r_factor_statistics This will output a plain text histograms for Rwork, Rfree and Rfree-Rwork. For example: *** phenix.r_factor_statistics 2.5 left_offset=0.2 right_offset=0.2 n_bins=5 Command used: phenix.r_factor_statistics 2.500 left_offset=0.200 right_offset=0.200 n_bins=5 Histogram of Rwork for models in PDB at resolution 2.30-2.70 A: 0.115 - 0.168 : 149 0.168 - 0.220 : 2998 0.220 - 0.273 : 1879 0.273 - 0.325 : 67 0.325 - 0.378 : 2 Histogram of Rfree for models in PDB at resolution 2.30-2.70 A: 0.146 - 0.210 : 116 0.210 - 0.274 : 3288 0.274 - 0.337 : 1664 0.337 - 0.401 : 26 0.401 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.30-2.70 A: 0.001 - 0.021 : 233 0.021 - 0.041 : 1412 0.041 - 0.060 : 2180 0.060 - 0.080 : 1045 0.080 - 0.100 : 225 Number of structures considered: 5095 *** and running it without arguments will take all structures. Pavel. On 2/25/10 2:01 PM, Simon Kolstoe wrote:

...

oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Simon

On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...
Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Phil Evans

9:30 a.m.

I do worry a bit about the fetishisation of R-factors, to the point where I get the impression that some people think that the object of refinement is minimisation of Rfree. The aim is to produce the "best" model consistent with the data, and the most important tool is looking at the maps on the graphics, in conjunction with validation tools analysing consistency with what we know about stereochemistry (Ramachandran, rotamers, bumps etc). Also is your model consistent with your initial experimental phased map, if available? The Rfree is a useful guide, but no more than that. My (admittedly superficial) experience of changing weights, freeR selection, resolution limits, NCS, etc in refinement is that often you can spend some time doing this, maybe improve the R-factors a bit, but end up with a model which is indistinguishable from the previous model when superimposed on it. I'm fed up with referees telling me my structure must be wrong because the Rfree is "too high" (and I haven't followed their particular pet protocol), when I know that it is essentially correct, and as good as I can make it. Phil On 26 Feb 2010, at 01:49, Pavel Afonine wrote:

...

Hi Simon,

the next available PHENIX nightly build will have this command:

phenix.r_factor_statistics

This will output a plain text histograms for Rwork, Rfree and Rfree-Rwork. For example:

*** phenix.r_factor_statistics 2.5 left_offset=0.2 right_offset=0.2 n_bins=5 Command used:

phenix.r_factor_statistics 2.500 left_offset=0.200 right_offset=0.200 n_bins=5

Histogram of Rwork for models in PDB at resolution 2.30-2.70 A: 0.115 - 0.168 : 149 0.168 - 0.220 : 2998 0.220 - 0.273 : 1879 0.273 - 0.325 : 67 0.325 - 0.378 : 2 Histogram of Rfree for models in PDB at resolution 2.30-2.70 A: 0.146 - 0.210 : 116 0.210 - 0.274 : 3288 0.274 - 0.337 : 1664 0.337 - 0.401 : 26 0.401 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.30-2.70 A: 0.001 - 0.021 : 233 0.021 - 0.041 : 1412 0.041 - 0.060 : 2180 0.060 - 0.080 : 1045 0.080 - 0.100 : 225 Number of structures considered: 5095 ***

and running it without arguments will take all structures.

Pavel.

On 2/25/10 2:01 PM, Simon Kolstoe wrote:

...
oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Simon

On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...
Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

4:03 p.m.

Hi Phil, yes, I absolutely agree with you, no fetishisation! One can easily abuse any tool, any measure. And a complete picture about a model quality stems from model geometry, model to data fit, local map quality and so on, and not only from a single value, Rfree for instance. A comparison tool is useful though. If you tell me you refined a structure at 0.7A resolution, and the Rfree is 15% it will definitely indicate a problem. In such a case it is a good idea to know this and 1) either do some more effort to improve your structure or 2) explain that you did your best and nothing else can be done to make the model better. Pavel. On 2/26/10 1:30 AM, Phil Evans wrote:

...

I do worry a bit about the fetishisation of R-factors, to the point where I get the impression that some people think that the object of refinement is minimisation of Rfree. The aim is to produce the "best" model consistent with the data, and the most important tool is looking at the maps on the graphics, in conjunction with validation tools analysing consistency with what we know about stereochemistry (Ramachandran, rotamers, bumps etc). Also is your model consistent with your initial experimental phased map, if available? The Rfree is a useful guide, but no more than that.

My (admittedly superficial) experience of changing weights, freeR selection, resolution limits, NCS, etc in refinement is that often you can spend some time doing this, maybe improve the R-factors a bit, but end up with a model which is indistinguishable from the previous model when superimposed on it.

I'm fed up with referees telling me my structure must be wrong because the Rfree is "too high" (and I haven't followed their particular pet protocol), when I know that it is essentially correct, and as good as I can make it.

Phil

On 26 Feb 2010, at 01:49, Pavel Afonine wrote:

...
Hi Simon,

the next available PHENIX nightly build will have this command:

phenix.r_factor_statistics

This will output a plain text histograms for Rwork, Rfree and Rfree-Rwork. For example:

*** phenix.r_factor_statistics 2.5 left_offset=0.2 right_offset=0.2 n_bins=5 Command used:

phenix.r_factor_statistics 2.500 left_offset=0.200 right_offset=0.200 n_bins=5

Histogram of Rwork for models in PDB at resolution 2.30-2.70 A: 0.115 - 0.168 : 149 0.168 - 0.220 : 2998 0.220 - 0.273 : 1879 0.273 - 0.325 : 67 0.325 - 0.378 : 2 Histogram of Rfree for models in PDB at resolution 2.30-2.70 A: 0.146 - 0.210 : 116 0.210 - 0.274 : 3288 0.274 - 0.337 : 1664 0.337 - 0.401 : 26 0.401 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.30-2.70 A: 0.001 - 0.021 : 233 0.021 - 0.041 : 1412 0.041 - 0.060 : 2180 0.060 - 0.080 : 1045 0.080 - 0.100 : 225 Number of structures considered: 5095 ***

and running it without arguments will take all structures.

Pavel.

On 2/25/10 2:01 PM, Simon Kolstoe wrote:

...
oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Simon

On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...
Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

> Hi everyone, > I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). > Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful. > > Thanks in advance, > > Young-Jin > > > _______________________________________________ > phenixbb mailing list > [email protected] > http://phenix-online.org/mailman/listinfo/phenixbb > > _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Maia Cherney

5:25 p.m.

I agree with Phil, I had a data set with a large gap between R factors, so I collected a dataset from another crystal. I got a small gap (3%) between R factors, but there was no difference between the structures. Maia Phil Evans wrote:

...

I do worry a bit about the fetishisation of R-factors, to the point where I get the impression that some people think that the object of refinement is minimisation of Rfree. The aim is to produce the "best" model consistent with the data, and the most important tool is looking at the maps on the graphics, in conjunction with validation tools analysing consistency with what we know about stereochemistry (Ramachandran, rotamers, bumps etc). Also is your model consistent with your initial experimental phased map, if available? The Rfree is a useful guide, but no more than that.

My (admittedly superficial) experience of changing weights, freeR selection, resolution limits, NCS, etc in refinement is that often you can spend some time doing this, maybe improve the R-factors a bit, but end up with a model which is indistinguishable from the previous model when superimposed on it.

I'm fed up with referees telling me my structure must be wrong because the Rfree is "too high" (and I haven't followed their particular pet protocol), when I know that it is essentially correct, and as good as I can make it.

Phil

On 26 Feb 2010, at 01:49, Pavel Afonine wrote:

...
Hi Simon,

the next available PHENIX nightly build will have this command:

phenix.r_factor_statistics

This will output a plain text histograms for Rwork, Rfree and Rfree-Rwork. For example:

*** phenix.r_factor_statistics 2.5 left_offset=0.2 right_offset=0.2 n_bins=5 Command used:

phenix.r_factor_statistics 2.500 left_offset=0.200 right_offset=0.200 n_bins=5

Histogram of Rwork for models in PDB at resolution 2.30-2.70 A: 0.115 - 0.168 : 149 0.168 - 0.220 : 2998 0.220 - 0.273 : 1879 0.273 - 0.325 : 67 0.325 - 0.378 : 2 Histogram of Rfree for models in PDB at resolution 2.30-2.70 A: 0.146 - 0.210 : 116 0.210 - 0.274 : 3288 0.274 - 0.337 : 1664 0.337 - 0.401 : 26 0.401 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.30-2.70 A: 0.001 - 0.021 : 233 0.021 - 0.041 : 1412 0.041 - 0.060 : 2180 0.060 - 0.080 : 1045 0.080 - 0.100 : 225 Number of structures considered: 5095 ***

and running it without arguments will take all structures.

Pavel.

On 2/25/10 2:01 PM, Simon Kolstoe wrote:

...
oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Simon

On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...
Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

> Hi everyone, > I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). > Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful. > > Thanks in advance, > > Young-Jin > > > _______________________________________________ > phenixbb mailing list > [email protected] > http://phenix-online.org/mailman/listinfo/phenixbb > > _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Felix Frolow

8:18 p.m.

Yea Phil, You have got them I was accused several years ago for over-fitting Rfree !!!!!!! The bad thing is that to publish you have to avoid arguing with these post-crystallography era referees. Today they ask meaningless question " I am novice to crystallography, teach me how to minimize Rfree" Tomorrow they referee you papers and make meaningless remarks about gap between R and Rfree " Your R factor is 17% and Rfree is 24%, it rase question" geeeeek!!!!!! what question????? Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: [email protected] Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Feb 26, 2010, at 11:30 , Phil Evans wrote:

...

I do worry a bit about the fetishisation of R-factors, to the point where I get the impression that some people think that the object of refinement is minimisation of Rfree. The aim is to produce the "best" model consistent with the data, and the most important tool is looking at the maps on the graphics, in conjunction with validation tools analysing consistency with what we know about stereochemistry (Ramachandran, rotamers, bumps etc). Also is your model consistent with your initial experimental phased map, if available? The Rfree is a useful guide, but no more than that.

My (admittedly superficial) experience of changing weights, freeR selection, resolution limits, NCS, etc in refinement is that often you can spend some time doing this, maybe improve the R-factors a bit, but end up with a model which is indistinguishable from the previous model when superimposed on it.

I'm fed up with referees telling me my structure must be wrong because the Rfree is "too high" (and I haven't followed their particular pet protocol), when I know that it is essentially correct, and as good as I can make it.

Phil

On 26 Feb 2010, at 01:49, Pavel Afonine wrote:

...
Hi Simon,

the next available PHENIX nightly build will have this command:

phenix.r_factor_statistics

This will output a plain text histograms for Rwork, Rfree and Rfree-Rwork. For example:

*** phenix.r_factor_statistics 2.5 left_offset=0.2 right_offset=0.2 n_bins=5 Command used:

phenix.r_factor_statistics 2.500 left_offset=0.200 right_offset=0.200 n_bins=5

Histogram of Rwork for models in PDB at resolution 2.30-2.70 A: 0.115 - 0.168 : 149 0.168 - 0.220 : 2998 0.220 - 0.273 : 1879 0.273 - 0.325 : 67 0.325 - 0.378 : 2 Histogram of Rfree for models in PDB at resolution 2.30-2.70 A: 0.146 - 0.210 : 116 0.210 - 0.274 : 3288 0.274 - 0.337 : 1664 0.337 - 0.401 : 26 0.401 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.30-2.70 A: 0.001 - 0.021 : 233 0.021 - 0.041 : 1412 0.041 - 0.060 : 2180 0.060 - 0.080 : 1045 0.080 - 0.100 : 225 Number of structures considered: 5095 ***

and running it without arguments will take all structures.

Pavel.

On 2/25/10 2:01 PM, Simon Kolstoe wrote:

...
oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Simon

On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...
Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote: > Hi everyone, > I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). > Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful. > > Thanks in advance, > > Young-Jin > > > _______________________________________________ > phenixbb mailing list > [email protected] > http://phenix-online.org/mailman/listinfo/phenixbb > _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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Schubert, Carsten [PRDUS]

2:18 p.m.

I second that, please add pdf, png or ps output as an option. In phenix the GUI is a choice, not a prerequisite to using the suite. I would welcome it if further development of the suite would adhere to this philosophy. Cheers, Carsten

...

-----Original Message----- From: [email protected] [mailto:phenixbb- [email protected]] On Behalf Of Simon Kolstoe Sent: Thursday, February 25, 2010 5:01 PM To: PHENIX user mailing list Subject: Re: [phenixbb] Gap between R and Rfree

oh yes please Pavel (command line polygon/statistics overview) - could the output be a pdf or ps rather than just starting up the GUI?

Simon

On 25 Feb 2010, at 19:39, Pavel Afonine wrote:

...
Hi Francis,

in PHENIX GUI: "Validation" -> "PDB Statistics Overview"

or:

in PHENIX GUI: "Validation" -> "POLYGON"

The underlying idea is published here:

Crystallographic model quality at a glance. L. Urzhumtseva, P. V. Afonine, P. D. Adams, A. Urzhumtsev Acta Cryst. D65, 297-300 (2009)

I can add a command line version if anyone is interested.

Pavel.

On 2/25/10 9:45 AM, Francis E Reyes wrote:

...
Pavel

What phenix utility produces such information? Thx

FR

On Feb 25, 2010, at 10:43 AM, Pavel Afonine wrote:

...
Hi Young-Jin,

the distribution of R-factors for all structures in PDB refined at resolutions between 2.4 and 2.6 A is following:

Histogram of Rwork for all model in PDB at resolution 2.40-2.60: 0.115 - 0.141 : 5 0.141 - 0.168 : 69 0.168 - 0.194 : 414 0.194 - 0.220 : 955 <<<<< your structure 0.220 - 0.246 : 695 0.246 - 0.273 : 153 0.273 - 0.299 : 25 0.299 - 0.325 : 5 0.325 - 0.352 : 0 0.352 - 0.378 : 1 Histogram of Rfree for all model in PDB at resolution 2.40-2.60: 0.146 - 0.178 : 3 0.178 - 0.210 : 41 0.210 - 0.242 : 404 0.242 - 0.274 : 1104 0.274 - 0.305 : 653 <<<<< your structure 0.305 - 0.337 : 108 0.337 - 0.369 : 8 0.369 - 0.401 : 0 0.401 - 0.433 : 0 0.433 - 0.465 : 1 Histogram of Rfree-Rwork for all model in PDB at resolution 2.40-2.60: 0.002 - 0.012 : 28 0.012 - 0.022 : 83 0.022 - 0.031 : 232 0.031 - 0.041 : 430 0.041 - 0.051 : 458 0.051 - 0.061 : 493 0.061 - 0.071 : 298 0.071 - 0.080 : 186 0.080 - 0.090 : 85 <<<<< your structure 0.090 - 0.100 : 29

which I interpret as your Rwork is most likely good as well as Rfree, but the gap Rfree-Rwork is too large meaning possible overfitting.

A few tips: - if you have NCS - use it in refinement; - optimize refinement target weights: a) automatically: "optimize_wxc=true optimize_wxu=true", or b) manually using wxc_scale and wxu_scale parameters; - make sure you use the latest PHENIX version.

Let me now if you have any questions.

Pavel.

On 2/25/10 9:17 AM, Young-Jin Cho wrote:

...
Hi everyone, I recently got a diffraction data and am trying to refine it. The question is whatever I did the gap between R and Rfree stay far away: .2140/.2982 around 2.5 A resolution. (water added and isotropic(individual_adp) and so on). Although I am redoing with many different approaches, if anyone can give me any comments or suggestions, it would be helpful.

Thanks in advance,

Young-Jin

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Maia Cherney

28 Feb 28 Feb

6:01 p.m.

New subject: magnesium

Hi, I have a ligand (ADP) has a coordinated Mg2+ ion. The distance between Mg and each of the 6 ligands should be around 2.1 A. Does the program have the proper restraints for this distance (Mg2+-to-O) or I should manually make these restraints? Maia

Pavel Afonine

6:19 p.m.

New subject: magnesium

Hi Maia, run phenix.metal_coordination model.pdb which will create elbow.edits file containing restraints definitions for Mg - check them and edit if necessary to match your expectations. Then, next time you run phenix.refine, just give this elbow.edits file at input: phenix.refine model.pdb data.mtz other_parameters elbow.edits I presume it can all be done from PHENIX GUI as well. Pavel. On 2/28/10 10:01 AM, Maia Cherney wrote:

...

Hi, I have a ligand (ADP) has a coordinated Mg2+ ion. The distance between Mg and each of the 6 ligands should be around 2.1 A. Does the program have the proper restraints for this distance (Mg2+-to-O) or I should manually make these restraints?

Maia _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Maia Cherney

14 Mar 14 Mar

12:24 a.m.

New subject: magnesium

Hi Pavel, does the program take into account that the Mg has 2+ charge (two electrons are missing)? I get negative density around Mg ion. Is it because of the lower occupancy or is it because the charge is not taken into account? Does the ion has a different name from neutral MG? like MG2+ ? I got the restraints for the coordination in the elbow.edits, but the Mg ion still has the same name MG. Maia Pavel Afonine wrote:

...

Hi Maia,

run

phenix.metal_coordination model.pdb

which will create elbow.edits file containing restraints definitions for Mg - check them and edit if necessary to match your expectations.

Then, next time you run phenix.refine, just give this elbow.edits file at input:

phenix.refine model.pdb data.mtz other_parameters elbow.edits

I presume it can all be done from PHENIX GUI as well.

Pavel.

On 2/28/10 10:01 AM, Maia Cherney wrote:

...
Hi, I have a ligand (ADP) has a coordinated Mg2+ ion. The distance between Mg and each of the 6 ligands should be around 2.1 A. Does the program have the proper restraints for this distance (Mg2+-to-O) or I should manually make these restraints?

Maia _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

George Phillips

12:28 a.m.

New subject: magnesium

Pavel, I second Maia's 'request' to get proper scattering factors into Phenix for metal ions! Metallobiochemists need love, too. George George N. Phillips, Jr., Ph.D. Professor of Biochemistry and of Computer Sciences University of Wisconsin-Madison 433 Babcock Dr. Madison, Wi 53706 Phone/FAX (608) 263-6142 On Mar 13, 2010, at 6:24 PM, Maia Cherney wrote:

...

Hi Pavel,

does the program take into account that the Mg has 2+ charge (two electrons are missing)? I get negative density around Mg ion. Is it because of the lower occupancy or is it because the charge is not taken into account? Does the ion has a different name from neutral MG? like MG2+ ? I got the restraints for the coordination in the elbow.edits, but the Mg ion still has the same name MG.

Maia

Pavel Afonine wrote:

...
Hi Maia,

run

phenix.metal_coordination model.pdb

which will create elbow.edits file containing restraints definitions for Mg - check them and edit if necessary to match your expectations.

Then, next time you run phenix.refine, just give this elbow.edits file at input:

phenix.refine model.pdb data.mtz other_parameters elbow.edits

I presume it can all be done from PHENIX GUI as well.

Pavel.

On 2/28/10 10:01 AM, Maia Cherney wrote:

...
Hi, I have a ligand (ADP) has a coordinated Mg2+ ion. The distance between Mg and each of the 6 ligands should be around 2.1 A. Does the program have the proper restraints for this distance (Mg2+-to-O) or I should manually make these restraints?

Maia _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Ralf W. Grosse-Kunstleve

12:36 a.m.

New subject: magnesium

...

I second Maia's 'request' to get proper scattering factors into Phenix for metal ions! Metallobiochemists need love, too.

I'll try to fix this next week. Ralf

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26 comments

13 participants

participants (13)

Felix Frolow
Francis E Reyes
George Phillips
Kendall Nettles
Maia Cherney
Nathaniel Echols
Pavel Afonine
Phil Evans
Phil Jeffrey
Ralf W. Grosse-Kunstleve
Schubert, Carsten [PRDUS]
Simon Kolstoe
Young-Jin Cho