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

Just got back from my afternoon walk... If you are insistent on placating the unruly mob and still want to publish an Rfree for real space refinement here is reasonably efficient way to avoid bias. Calculate your MAPcalc by first calculating Fcalc and follow up by a Fourier synthesis. Before the synthesis replace all the test set Fcalc equivalents with their Fobs counterparts. F's are complex numbers here so you are putting in both the observed amplitude and phase. When you compare this map to the observed map the model will never be penalized for disagreeing about test set reflections and is free to do whatever it wants. This does not really add any more time to the calculation since you have to calculate Fcalcs anyway if you are going to report R values. It also allows you to use the experimental map w/o ever having to modify it. As a general rule I think experimental results should be kept in read-only files during interpretation. Dale Tronrud P.S. I still think the whole idea of Rfree in real-space refinement is not a good one. -------- Forwarded Message -------- Subject: Re: [phenixbb] phenix.map_to_model input mtz file failure --caution on using map_to_model with X-ray dataDate: Tue, 13 Jun 2017 14:40:57 -0700 From: Dale Tronrud To: [email protected] On 6/13/2017 12:30 PM, Edward A. Berry wrote:

Thanks, Pavel, I really appreciate your taking the time to generate the example.

While I agree with Tim and Ian that refinement to convergence should remove the bias making it perhaps not a serious problem, my question was in fact whether there is any bias immediately after the refinement.

I will need to study this example a bit, but one thing I notice is that you are doing exactly what I was guessing, comparing Rfree after real-space refinement with and without using the free set. Then, I still think, we

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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).

Of course the model is refined as though the test set Fourier components were equal to zero. In reciprocal space refinement when you leave a reflection out of the "sum over all reflections" when calculating the difference map you are saying that you have no opinion about the amplitude of that reflection. When you calculate a real space map from Fourier coefficients you can't not have an opinion, i.e. you can't leave a term out of the sum you can only set that term to zero. If your model produces a prediction for that term which is not equal to zero it will be penalized. (If you set that term to Fcalc you tie your model to its starting point. To unbias you would have to calculate a new map with current Fcalc's for every iteration of the model, but this method would not take into account the neighborhood correlation present in experimental maps.) What this means is that Rfree is not a meaningful stat for assessing overfitting of real space refinement. This is hardly a surprise. A test of a refinement protocol has to be based on the mathematics of that protocol, not the protocol you happened to have used yesterday. If you want an unbiased estimate of the quality of a real space refinement you have to leave out a region of the map and then see how well the model fits that region. This is harder to do in an automated fashion and there will be a lot of caveats about your results (e.g. you know about the ability to fit one region but does that generalize to other areas?). If you recall there are a lot of caveats about Rfree too - we have just stopped worrying about them. (e.g. low resolution vrs high resolution reflections, choosing based on shells or randomly, what to do about ncs...) I think you should consider yourself on the wrong track if you come up with a statistical test, but haven't given any thought to the actual experiment that produced your map. Dale Tronrud

Things I need to look at- What are R and R-free for the original refined model What are R and R-free after shaking (did RSR lower R but not Rfree, or did it raise Rfree? What if RSR is done using a map made with fill-in strategy?

Ed

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

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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:

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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:

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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:

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​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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