[3dem] pdb for near-atomic resolution EM maps

Sjors Scheres scheres at mrc-lmb.cam.ac.uk
Tue Dec 2 00:34:03 PST 2014

Hi Irina,
This is an excellent question that we really should discuss as a
community. I guess that while we have not reached a "standard", it is most
important that you describe in detail what you did to obtain your model in
the methods section of your paper. This should also reflect the confidence
(or lack of it) that you have in the different parts of your model. 4.3A
is a "frustating" resolution in some ways: as you mention you start to see
many side chains and that gets you excited, but still a lot of ambiguity
remains when you build an atomic model. We had a similar situation with
gamma-secretase. We did the following. Where a decent homology model
existed, we made a model with side chains (which I guess will still have
errors). Where we were less sure (in the trans-membrane part), we built
only a backbone model. Our resolution of 4.5A meant we were not done yet,
and we are currently investing a lot of time in making the reconstruction
good enough to propose a reliable atomic model.

BTW: Masahide, R_free measures overfitting of the model. It does not
measure errors in it. For that, we have found Ramachandran plots and other
geometry-reporters to be more useful. However, at resolutions that stay
below 4A, it will probably remain very difficult not to make mistakes.

> Hi Irina,
>> I would like to deposit a near-atomic resolution map (4.3A) and the
>> corresponding atomic model to the respective databases. Given recent
>> discussions about overinterpretation of the data I am wondering what
>> would be the best way to proceed.
>> Indeed, some bulky side chains are clearly visible and could be placed
>> with confidence, in particular in certain regions of the map (according
>> to ResMap local resolution ranges from 3.5A to above 6A), but most of
>> them not.
> If you look into the PDB database, there are plenty of "atomic structures"
> build based on 4-6A resolution X-ray crystallographic data. Since phase
> information derived from X-ray is not as reliable as those from cryo-EM at
> this resolution range, those atomic models often turned out to be wrong.
> On the other hand, we (electron microscopists) should try to extract as
> much data as possible and try to interpret the density map, with caution.
> Therefore, in my opinion, it is user's responsibility to check the
> validity of the "model" deposited to PDB.
> To make this user's validation easier, a combination of EM density map
> (EMDB) and corresponding atomic coordinate (PDB) should be deposited. In
> the future, it would be desirable to develop a cryo-EM version of  Rfree
> (already exists?).
>> And even the criteria of "visibility" and "confidence" are subjective.
>> The protein complex is of big pharmaceutical interest and I suppose that
>> if the complete pdb with all the side chains is deposited, it may be
>> downloaded and used for example for drug design, as a kind of "absolute
>> truth", which might be dangerous. However, deposit only the c-alpha is
>> probably too restrictive. Another option would be to deposit only the
>> side chains we are "sure about", but again, it's subjective, and anyway
>> we can only be "sure" at the given resoluition. So what would be the
>> most honest and unbiased way to share these results with the scientific
>> community, in particular with biologists who are not necessarily aware
>> of the overinterpretation/overfitting/validation/etc issues in our field
>> but who use our results to design their experiments?
> ---------------------------------------------------------------
> Masahide Kikkawa, M.D., Ph. D.
> Professor
> Department of Cell Biology & Anatomy
> Graduate School of Medicine
> The University of Tokyo
> http://structure.m.u-tokyo.ac.jp/
> tel: +81-3-5841-3338
> fax: +81-3-5841-3339
> ---------------------------------------------------------------
> _______________________________________________
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Sjors Scheres
MRC Laboratory of Molecular Biology
Francis Crick Avenue, Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44 (0)1223 267061

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