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Dear Ariel,<br>
<br>
I think there is a major confusion about what is modeling and what are experimentally determined constrains. Rotamer libraries are derived from experimental data, just like the rest of geometrical parameters. Low resolution refinement and data interpretation
for that matter heavily rely on rotamer libraries. I mentioned MD in my previous message as something that seriously dedicated people might want to try using the structure that
<b>has been deposited</b> to PDB or elsewhere <b>as</b> <b>a main chain</b> for which side chains have just been recreated. I
<b>never suggested</b> depositing MD simulated data to PDB. <br>
<br>
In principle, side chains at 4-5... A resolution are similar to hydrogen atoms at ~1-1.2 A in X-ray. You can't really see most of them, but you know they are there and they can be easily built using geometry (and the crystallographic community has annual discussions
of what to do with H atoms - deposit or not). Conformational space of most side chains is much greater than that of hydrogens, but rotamer libraries help us to reduce it to just a few conformations and this is what ALL the refinement programs use at low resolution.<br>
<br>
On the other hand, I am a little disturbed by your remark that you chose to deposit a structure that clearly does not agree with your experimental map. Shouldn't it be a C-alpha trace or something similarly less precise than a complete atomic model?<br>
<br>
Best wishes,<br>
<br>
Petr<br>
<br>
<div class="moz-cite-prefix">On 12/02/2014 10:38 AM, Ariel Blocker wrote:<br>
</div>
<blockquote cite="mid:99E50CF1-DEEE-486D-A8DA-F33B3DA79429@bristol.ac.uk" type="cite">
Dear Irina,
<div><span class="Apple-tab-span" style="white-space:pre"></span>I agree with the other replies that this is a key question for the EM community now.</div>
<div><span class="Apple-tab-span" style="white-space:pre"></span>We recently had a similar "cas de conscience”, but with a crystal structure docking to an even much lower resolution map, where it is absolutely obvious that none of the side chains can be accurate
(and maybe the polypeptide chain fold isn’t vaguely right in places!). We decided to deposit the pseudoatomic model in the pdb anyway, partly because it is what EMDB recommend, partly because our journal editor also said he felt it was appropriate but also
partly because we felt that if we didn’t nobody could independently reassess/test/use our work in the future. Now, in theory, all those accessing such files should be able to figure out for themselves how accurate the models are but, in reality, many I feared
would be fooled into taking their visual accuracy for granted. So, we decided to put a “resolution health warning” as a note in the deposition, which I requested would be available upfront to the downloaders. And, of course, we also discussed the crudeness
of the resolution in the paper. </div>
<div><span class="Apple-tab-span" style="white-space:pre"></span>Regarding the rapidly increasing numbers of 4-5 Å resolution cryoEM maps, I think we should also caution reviewers and users about relying on the rotamer libraries and MD (rather than additional
ED map refinement, where possible, which should be done by authors) to improve the pseudoatmoic models. That is mixing modelling with experimental data in ways that, as far as I know, are not properly experimentally investigated/validated yet (but evidently
need to be in future), and where there is therefore again serious concern about overinterpretation. So, if we chose do it, it should also be explicit in the deposition and paper.</div>
<div><span class="Apple-tab-span" style="white-space:pre"></span>Best,</div>
<div><span class="Apple-tab-span" style="white-space:pre"></span>Ariel.</div>
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<div>
<div>On 2 Dec 2014, at 09:03, Petr Leiman <<a moz-do-not-send="true" href="mailto:petr.leiman@epfl.ch">petr.leiman@epfl.ch</a>> wrote:</div>
<br class="Apple-interchange-newline">
<blockquote type="cite">
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<br>
On 12/02/2014 08:43 AM, Irina Gutsche wrote:<br>
<blockquote type="cite"><br>
Dear all,<br>
<br>
I would like to deposit a near-atomic resolution map (4.3A) and the<span class="Apple-converted-space"> </span><br>
corresponding atomic model to the respective databases. Given recent<span class="Apple-converted-space"> </span><br>
discussions about overinterpretation of the data I am wondering what<span class="Apple-converted-space"> </span><br>
would be the best way to proceed.<br>
Indeed, some bulky side chains are clearly visible and could be placed<span class="Apple-converted-space"> </span><br>
with confidence, in particular in certain regions of the map<span class="Apple-converted-space"> </span><br>
(according to ResMap local resolution ranges from 3.5A to above 6A),<span class="Apple-converted-space"> </span><br>
but most of them not. And even the criteria of "visibility" and<span class="Apple-converted-space"> </span><br>
"confidence" are subjective. The protein complex is of big<span class="Apple-converted-space"> </span><br>
pharmaceutical interest and I suppose that if the complete pdb with<span class="Apple-converted-space"> </span><br>
all the side chains is deposited, it may be downloaded and used for<span class="Apple-converted-space"> </span><br>
example for drug design, as a kind of "absolute truth", which might be<span class="Apple-converted-space"> </span><br>
dangerous. However, deposit only the c-alpha is probably too<span class="Apple-converted-space"> </span><br>
restrictive. Another option would be to deposit only the side chains<span class="Apple-converted-space"> </span><br>
we are "sure about", but again, it's subjective, and anyway we can<span class="Apple-converted-space"> </span><br>
only be "sure" at the given resoluition. So what would be the most<span class="Apple-converted-space"> </span><br>
honest and unbiased way to share these results with the scientific<span class="Apple-converted-space"> </span><br>
community, in particular with biologists who are not necessarily aware<span class="Apple-converted-space"> </span><br>
of the overinterpretation/overfitting/validation/etc issues in our<span class="Apple-converted-space"> </span><br>
field but who use our results to design their experiments?<br>
</blockquote>
<br>
This kind of topic is discussed at crystallographic BBs at length about<span class="Apple-converted-space"> </span><br>
once a year for as long as those boards exit. There, however, people<span class="Apple-converted-space"> </span><br>
discuss what to do with side chains for which there is no density. Build<span class="Apple-converted-space"> </span><br>
them or truncate at C-alpha or C-beta or make them zero occupancy or let<span class="Apple-converted-space"> </span><br>
the refinement take care of the problem (it will elevate the B factor to<span class="Apple-converted-space"> </span><br>
hundred(s) of A^2). These discussions are endless for the exact same<span class="Apple-converted-space"> </span><br>
reasons that Irina has brought up.<br>
<br>
In the case of 3.5-6 A resolution structure, the experimental error of<span class="Apple-converted-space"> </span><br>
atomic positions is so high that you build your side chains based on the<span class="Apple-converted-space"> </span><br>
rotamer database, but not on the density. There is also an uncertainty<span class="Apple-converted-space"> </span><br>
(e.g. error) associated with placing the main chain, but let's forget<span class="Apple-converted-space"> </span><br>
about this for a second. My point: for these "almost atomic<span class="Apple-converted-space"> </span><br>
interpretation" datasets, the information contained in the main chain<span class="Apple-converted-space"> </span><br>
that is placed into the density to the best of our ability is all that<span class="Apple-converted-space"> </span><br>
is required to recreate the complete atomic model. For simple<span class="Apple-converted-space"> </span><br>
interpretations and ribbon diagrams, the main chain information is<span class="Apple-converted-space"> </span><br>
sufficient. But people who want to design drugs will have to look into<span class="Apple-converted-space"> </span><br>
the model a little more. They will have to rebuild all the side chains<span class="Apple-converted-space"> </span><br>
(they will rely on the rotamer library for doing so - exactly what we do<span class="Apple-converted-space"> </span><br>
now) and perform MD and/or additional refinement. And of course, the<span class="Apple-converted-space"> </span><br>
main chain model should contain all the side chains for which there is<span class="Apple-converted-space"> </span><br>
good experimental density.<br>
<br>
Best wishes,<br>
<br>
Petr<br>
<br>
<blockquote type="cite"><br>
Thank you very much for your all ideas and suggestions,<br>
<br>
Best regards,<br>
<br>
Irina Gutsche<br>
CNRS, Grenoble,<br>
France<br>
<br>
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<br>
--<span class="Apple-converted-space"> </span><br>
Petr Leiman<br>
Laboratory of Structural Biology and Biophysics<br>
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<br>
<pre class="moz-signature" cols="72">--
Petr Leiman
Laboratory of Structural Biology and Biophysics
<a class="moz-txt-link-freetext" href="http://lbbs.epfl.ch">http://lbbs.epfl.ch</a>
EPFL
BSP-415
CH-1015 Lausanne
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Mobile: +41 79 538 7647
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