Re: [phenixbb] phenix.map_to_model input mtz file failure --caution on using map_to_model with X-ray data

How about this for an explanation of why setting Fcalc to zero doesn't hurt so much? Lets assume that Fcalc is correlated with its corresponding Fobs. (This is reasonable since your R values are 20 to 30%.) I presume you are refining against some sort of (2Fo-Fc, PhiC) map. If you put in the real Fc you will get amplitudes that are roughly equal to (Fo, PhiC) since the Fc roughly cancels out one of the Fo. For reflections where Fc is zeroed out the coefficient will be, roughly, (2Fo, PhiC). 2Fo is still pretty strongly correlated to Fo. Certainly large Fo's will produce large 2Fo's and small Fo's will produce small 2Fo's. A reflection with twice the amplitude but the same phase will have peaks and valleys in exactly the same place, so I wouldn't expect huge differences in the location of atoms. Now I have to ask exactly how this "set Fcalc to zero" works. The simple-minded thing would be to set Fc to zero in 2Fo-Fc and get 2Fo, but I don't think this is the best thing to do. The logic for "(2Fo-Fc,Phic)" is that this is a good estimate of the "true" Fourier coefficient given that you know the Fo amplitude and a (Fc,Phic) from a model. If you say, instead, that you know only the Fo amplitude and Phic, you find that the centroid of that probability distribution is (Fo, Phic). Of course it is a little odd to say that you have a high confidence in Phic but no confidence in Fc... Since they are so tightly coupled I would rather assume I don't know either if I wanted to leave the test reflection out of the map calculation. In that case the entire coefficient has to be set to zero. Maybe that is what is being done here. Dale Tronrud On 6/18/2017 7:31 PM, Edward A. Berry wrote:

On 06/13/2017 02:15 PM, Pavel Afonine wrote:

...

...

The result is self-explicable and is inline with Tom's reply to Wei.

Thanks, Pavel. Yes, that is convincing. I filled in some of the other results I wanted, and they do not change the conclusion.

in case anyone is interested:

After shaking, R and Rfree are very high and not significantly different (values below).

RS Refinement, against any of the maps generated from the refined structure. greatly reduces both. (So R-free is NOT increasing due to free reflections being zero in the transform of the map). If free reflections are excluded in making the map then R-free is higher than Rwork.

Using fill-in resulted in an R-free gap similar to that with excluding reflections. R and R-free were both a little lower with fill-in, but they were lower by about the same amount when the free set was included, so this may be more the effect of filling in the truly missing reflections than filling the free reflections. And could be considered as Fc-Phic bias towards the excellent map from fully refined structure.

Refining the original refined model from the pdb by RSR (against its own maps) resulted in R, R-free increasing to the same values obtained starting with the shaken models. This is like the increase seen when you solve a structure with a high-resolution search model and refine against a low-resolution dataset.

Making the maps using Fobs and the Fc Phi-c from the shaken model rather than final refined model, which is more relevant for a crystallographer (in case any crystallographer should ignore the advice given already in this thread and use real-space-refine/map-to-model instead of autobuild) still gave a significant decrease in both R's, and a similar R-Rfree gap (or lack thereof) in all four cases. Fill-in gave higher R's this time (Fc-Phic bias toward the original shaken model?)

Specifically:

model R-work R-free Original fully refined PDB 0.171 0.221 from header

After dynamics shaking: 0.3923 0.3917

After RS refinement of shaken: Using all reflections 0.2409 0.2434 Using all, + Fc for missing 0.2352 0.2409 excluding free 0.2419 0.2731 Fc for free (and missing) 0.2387 0.2606

After RS refinement of 1f8t: Using all reflections 0.2421 0.2453 Using all, + Fc for missing 0.2425 0.2479 excluding free 0.2453 0.2742 Fc for free (and missing) 0.2393 0.2585

After RS refinement of shaken against map made from shaken: Using all reflections 0.2870 0.2915 Using all, + Fc for missing 0.3008 0.3034 excluding free 0.2917 0.3236 Fc for free (and missing) 0.3026 0.3290

It is surprising to me that making the map with fillin Fc from the refined model vs the bad starting model makes a significant difference, but making the map with Fc=0 doesn't seem to hurt.

On 06/13/2017 02:15 PM, Pavel Afonine wrote:

...

Hi Ed,

Including free-r reflections into map calculation and then using such map in real-space refinement of entire model will affect Rfree. Here is a simple example that illustrates my statement, step-by-step:

1) Get data and model from PDB:

phenix.fetch_pdb 1f8t --mtz

2) Compute two 2mFo-DFc maps: one includes all reflections the other one has no free-r terms:

phenix.python run.py 1f8t.{pdb,mtz}

This will create an MTZ file (map_coeffs.mtz) that contains Fourier map coefficients for both maps.

3) Shake model a bit:

phenix.dynamics 1f8t.pdb number_of_steps=500

4) Run real-space refinement using two maps:

phenix.real_space_refine map_coeffs.mtz 1f8t_shaken.pdb label="work,PHIwork" ncs_constraints=false output.file_name_prefix=work

phenix.real_space_refine map_coeffs.mtz 1f8t_shaken.pdb label="all,PHIall" ncs_constraints=false output.file_name_prefix=all

5) Compute R-factors using data and real-space refined models:

phenix.model_vs_data 1f8t.mtz all_real_space_refined.pdb r_work(re-computed) : 0.2419 r_free(re-computed) : 0.2441

phenix.model_vs_data 1f8t.mtz work_real_space_refined.pdb r_work(re-computed) : 0.2444 r_free(re-computed) : 0.2756

The result is self-explicable and is inline with Tom's reply to Wei.

All files necessary to reproduce calculations above are here: http://cci.lbl.gov/~afonine/tmp/

All the best, Pavel

On 6/8/17 10:05, Tim Gruene wrote:

...

Hi Ed,

including the 'free' reflections in the map for modelling does not taint the value of Rfree. That is a misconception that i s very persistent (as prejudice usually are). I believe it was Ian Tickle who formulated that when you simply refine long enough towards convergence, all reflections excluded from refinement will become independent, i.e. you can assign a new set for Rfree every time you refine, if you wish so.

This concept is the reason why Rcomplete (the "better" equivalent to Rfree for small data sets with < 10,000 unique reflections), introduced by Axel Brunger, works, as we could demonstrate in doi: 10.1073/pnas.1502136112

So nothing to worry about when including all reflections in map calculations.

Cheers, Tim

On Thursday, June 8, 2017 12:42:53 PM CEST Edward A. Berry wrote:

...

Hi, Tom, Please forgive what may be a silly question from an outsider who hasn't really kept up with the crystallography literature or even all the Phenix newsletters- What is the evidence that including the free set in real space refinement biases R-free of the resulting model? Is this Rfree also biased when map coefficients use "fill-in" for the excluded free reflections (and is that what phenix.remove_free_from_map does?).

My point is that literally excluding the free reflections, as opposed to substituting their values with Fc, will bias the free set toward grossly incorrect values (namely zero) and therefore greatly worsen R-free. Thus if the evidence for bias is that you get worse R-free when you exclude the free set, you have to think about how much of that difference results from bias towards the observed values (when the reflections are included) and how much is from bias towards zero (when the free set is excluded). (Again, I realize this may be all very well understood by the crystallography community and properly taken care of in phenix; I'm just asking for my own information) eab

On 06/08/2017 07:28 AM, Terwilliger, Thomas Charles wrote:

...

​Hi Wei,

I want to give a word of caution about how to use phenix.map_to_model on crystallographic data...The bottom line is you should remove the test set from your map coefficients before running phenix.map_to model on X-ray data. Here is why:

phenix.map_to_model uses real-space refinement, which is refinement against the map. If you supply map coefficients that include your test reflections, then you will be refining against data that is in your test set. This will make your Rfree invalid when you go back and refine your model against the original crystallographic data.

To remove the test set from your map coefficients you can use:

phenix.remove_free_from_map map_coeffs=my_map_coeffs.mtz free_in=my_data_file_with_freeR_flags.mtz mtz_out=my_map_coeffs_no_free.mtz

Also note that phenix.map_to_model uses a fixed map (it does not do density modification). Consequently for most crystallographic data at moderate resolution or higher phenix.autobuild is going to do much better than phenix.map_to_model.

All the best,

Tom T

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---------------------------- *From:*[email protected] on behalf [email protected] *Sent:* Tuesday, June 6, 2017 9:16 PM *To:* Terwilliger, Thomas Charles *Cc:*[email protected] *Subject:* Re:Re: [phenixbb] phenix.map_to_model input mtz file failure Dear Thomas, I use CAD to convert the labels from FDM->FWT, PHIDM->PHFWT, then submit this job again (without map_coeffs_labels=... ), and everything seems ok. Thank you very much for you help. Best!

-- Wei Ding P.O.Box 603 The Institute of Physics,Chinese Academy of Sciences Beijing,China 100190 Tel: +86-10-82649083

E-mail:[email protected] mailto:[email protected]

At 2017-06-07 10:32:14, "Terwilliger, Thomas Charles" wrote: Hi Wei,

I'm sorry for the trouble!

If you supply an MTZ file that has FWT,PHFWT or similar labels, then you can skip the "labels=...." statement and it should run.

Let me know if that does not work! All the best,

Tom T

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---------------------------------------------------------------------- ---------- *From:*[email protected] mailto:[email protected] mailto:[email protected]> on behalf of [email protected] mailto:[email protected] mailto:[email protected]> *Sent:* Tuesday, June 6, 2017 8:19 PM *To:*[email protected] mailto:[email protected] *Subject:* [phenixbb] phenix.map_to_model input mtz file failure Dear Phenix bb, I intend to build a initial model by phenix.map_to_model. And the command line is as follows: phenix.map_to_model_1.12rc0-2787 map_coeffs_file=../rep_dm.mtz map_coeffs_labels="'FP,SIGFP' 'PHIDM' 'FOMDM'" seq_file=../resolve.seq is_crystal=True use_sg_symmetry=True density_select=False truncate_at_d_min=True and the feedback like this: Sorry: No initial assignment made for map_coeffs. Labels used: FP,SIGFP PHIDM FOMDM. Available labels: ['PHIB', 'FOM', 'HLA,HLB,HLC,HLD', 'FP,SIGFP', 'PHIDM', 'FOMDM', 'FDM', 'HLADM,HLBDM,HLCDM,HLDDM'] NOTE: grouped labels like 'FP,SIGFP' must stay together, have commas, and have no spaces. If they come from an MTZ file, they must be in adjacent columns as well. Suggested labels to use: PHIDM FOMDM I try many other input format of map_coeffs_labels, such as map_coeffs_labels="FP,SIGFP PHIDM FOMDM" map_coeffs_labels=["FP,SIGFP PHIDM FOMDM"] ... ... but the result is the same. Dose anyone can tell me how to fix this problem? Thank a lot.

-- Wei Ding P.O.Box 603 The Institute of Physics,Chinese Academy of Sciences Beijing,China 100190 Tel: +86-10-82649083 E-mail:[email protected] mailto:[email protected]

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