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<div dir="ltr">There is certainly a variability in water positions. For example, with a very slight improvement in resolution (~0.1 A), the center of the peak shifts, often by more than an Angstrom. The same happens if you use different subsets of a larger
dataset (or, equivalently, if you look at the independent half maps, as already pointed out).... One of numerous reasons why validating water positions is necessary. </div>
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<blockquote type="cite">On Dec 11, 2019, at 4:01 PM, Pintilie, Greg <gregp@slac.stanford.edu> wrote:<br>
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I was just about to send a reference to our bioRxiv paper but Dmitry beat me to it - glad to see it referenced!</div>
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I should also point out we have a Chimera plugin that tries to make it easy to calculate the Q-scores for individual water atoms, which can give some idea as to how nice the bump is, and also to extract parts of the map for closer inspection along with waters
as in the attached image. Could be fun to try it after placing waters with Coot as has been mentioned:</div>
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<a href="https://cryoem.slac.stanford.edu/ncmi/resources/software/mapq">https://cryoem.slac.stanford.edu/ncmi/resources/software/mapq</a><br>
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We are also very interested in all the questions that were brought up - how do we know they're really waters and not random noise? For the maps of Apoferritin we looked at, the bumps in the CryoEM maps were the same or very close to the waters placed in the
X-ray structure, so that's a relief. (And they should be in both half maps indeed).</div>
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<span style="color: rgb(0, 0, 0); font-family: Calibri, Arial, Helvetica, sans-serif; font-size: 12pt;">Statistically-speaking, for X-ray structures if we plot radial distances (figure 8 in the paper), we see a sharp peak at 2.8Å for distances from water to
other polar atoms. If the distance is not quite right in the structure, watch out for the undowser...</span><br>
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<a href="https://www.biorxiv.org/content/10.1101/795161v1" id="LPNoLP820671">https://www.biorxiv.org/content/10.1101/795161v1</a><br>
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Interestingly, putting waters directly on peaks in cryoEM maps, the radial plots are a bit different, and the peaks are more diffuse... does this mean they are not really waters if they are not exactly 2.8Å away, or could it be the uncertainty in position due
to limited resolutions or variabilities in the structure. <span style="color: rgb(0, 0, 0); font-family: Calibri, Arial, Helvetica, sans-serif; font-size: 12pt;">Some other recent work has shown that even with X-ray, distances and ADPs for water molecules
can change if the structure changes: </span><a href="https://pubs.acs.org/doi/10.1021/jacs.9b06275" id="LPNoLP271638" style="font-family: Calibri, Arial, Helvetica, sans-serif; font-size: 12pt;">https://pubs.acs.org/doi/10.1021/jacs.9b06275</a></div>
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So, I think interesting questions remain about how to detect and interpret the waters especially in cryoEM, where the peaks seem to be there, and it is so tempting to put waters (or metal ions perhaps) in them.</div>
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Greg</div>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Dmitry Lyumkis <dlyumkis@salk.edu><br>
<b>Sent:</b> Wednesday, December 11, 2019 3:54 PM<br>
<b>To:</b> radu@mrc-lmb.cam.ac.uk <radu@mrc-lmb.cam.ac.uk><br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br>
<b>Subject:</b> Re: [3dem] Placing waters</font>
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<div class="" style="word-wrap:break-word">There is also a function in Coot to place waters into bumps of density (Other Modeling Tools —> Find Waters). But you still have to go through the map/model and delete ones that are not appropriate.
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<div class="">On that note, I have a broader question for the list of how one would validate the water placements? We have the advantage that we’re working with multiple high-resolution maps that are otherwise (nearly) identical, with the exception of bound
ligand. Therefore it is actually straightforward (albeit tedious) to cross-validate the waters using independently refined maps, even at borderline resolutions. However, this is not the case when you only have a single map with which to work. In such a case,
you have to look at the surrounding chemistry, consider the size of the bump of the density, perhaps use tools to quantify the resolvability of the bump. One example of the latter was recently posted on bioRxiv (<a href="https://www.biorxiv.org/content/10.1101/722991v2" class="">https://www.biorxiv.org/content/10.1101/722991v2</a>).
Coot also has a validation tool, which considers the B-factor the placed water, the closest contacts, etc. </div>
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<div class="">My question to the list is: does anyone have good suggestions for validating water molecules, especially at borderline resolutions? Is there a set standard? </div>
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<div class="">Dmitry </div>
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<div class="">On Dec 11, 2019, at 10:22 AM, <a href="mailto:radu@mrc-lmb.cam.ac.uk" class="">
radu@mrc-lmb.cam.ac.uk</a> wrote:</div>
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<div class="">Hi Stephen,<br class="">
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We use Coot+Phenix and they work beautifully, just like in the X-ray world :-)<br class="">
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Best wishes,<br class="">
Radu<br class="">
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<blockquote type="cite" class="">Dear All,<br class="">
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What programs do people use to place and refine waters in high<br class="">
resolution cryoEM maps ?<br class="">
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thanks<br class="">
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Stephen<br class="">
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--<br class="">
<br class="">
**********************************************************************<br class="">
Dr. Stephen Cusack, FRS<br class="">
Head of EMBL Grenoble<br class="">
Group leader in the structural biology of protein-RNA complexes and viral<br class="">
proteins<br class="">
**********************************************************************<br class="">
<br class="">
Email:<span class="x_Apple-tab-span" style="white-space:pre"> </span><a href="mailto:cusack@embl.fr" class="">cusack@embl.fr</a>,
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Website: <a href="http://www.embl.fr" class="">http://www.embl.fr</a><br class="">
Tel:<span class="x_Apple-tab-span" style="white-space:pre"> </span>(33) 4 76 20 7238 Secretary (33) 4 76 20 7123<br class="">
Fax: (33) 4 76 20 7199<br class="">
Postal address: EMBL Grenoble Outstation, 71 Avenue des Martyrs, CS 90181,<br class="">
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