Re: [phenixbb] anomalous difference map

-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 On 10/10/2014 8:24 AM, Scott Horowitz wrote:

So I have a followup question to this comment in the thread: what is the origin of this rule of thumb of 1 and 3 sigma that people quote for non-difference and difference maps that you can find all over the internet at various sites, but nobody references? I can't find any papers on it specifically, but it's quite possible I'm not digging far enough back in time. Can anyone give me a lead?

Thanks, Scott

These particular contour levels are just "rules of thumb" as you say. The mistake of referring to them as "sigmas" gives the illusion of some sort of statistical basis. They are simply rmsd's and have no mathematical justification for being used to assess the quality, significance, or accuracy of a ligand identification (as Pavel noted). The 3 rmds contour level for Fo-Fc style maps is particularly problematic. The rmsd of the difference map for a model in the early stages of refinement will be large while at the end it will be small. The same piece of density due to missing atoms will appear weak in the early map and strong later. Conversely the features visible in an early map will correspond to striking errors in the model while features in a final difference map that appear at exactly the same strength will reflect tiny changes. This lack of calibration results in a continual hunt for poorer and poorer water molecules because no matter how many you build there are still plenty of "3 sigma" peaks in the difference map. The paper by Lang, Holton, Fraser, & Alber, recommended by Pavel, discusses many of these issues. They show that the real standard deviation of electron density maps is much lower than these rmsd values. The problem isn't that density in a 2Fo-Fc style map less than 1 rmsd isn't statistically significant but that it is hard to distinguish between alternate models that all fit this density. Telling the difference between a partially occupied ligand and superimposed puddles of partially occupied water molecules is pretty tough, particularly when the ligand is, itself, superimposed on partially occupied water. To keep my focus on some sort of an absolute scale I always think of contour levels in terms of electrons/A^3. This calculation is also problematic as is described in Lang et al. Another tool I use is to leave out a fully occupied water molecule that I have confidence in. I can compare mystery density with this known control to see if I am puzzling over a fully occupied atom or something weaker. With a raw Fo-Fc map you can never be sure. This "1 and 3 sigma" business really needs to be stomped out. Dale Tronrud

On Wed, Sep 10, 2014 at 9:26 AM, Pavel Afonine wrote:

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Hi Charles,

I think the key here is what you call "no difference density peak". Sites may be partially occupied so rule of thumb for choosing contouring levels for 2mFo-DFc and mFo-DFc maps (1 and 3 sigma, correspondingly) may not be appropriate, for instance.

Pavel

On 9/10/14 6:10 AM, CPMAS Chen wrote:

Thanks, Nat.

If this is noise, why the anomalous or LLG peaks could be as high as 5 ~ 6 sigma?

Charles

On Tue, Sep 9, 2014 at 5:58 PM, Nathaniel Echols wrote:

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On Tue, Sep 9, 2014 at 8:40 AM, CPMAS Chen wrote:

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Is it possible that I have anomalous and LLG peak, but I have no difference density peak? In this case, is that because the model I have is not good enough or the diffraction data at this site of the model is missing?

You should at a minimum see > 1sigma density in the 2mFo-DFc map, and if the site is unmodeled (or modeled as a water) you should see an mFo-DFc peak as well. If neither of these applies, the anomalous and LLG peaks are probably just noise.

-Nat

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Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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