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<p>Hello Francesca,</p>
<p><br>
</p>
<p>To determine whether you are seeing over-sharpening or something else, compare the unfiltered/unsharpened map and the post-processed map. Their respective filenames end in _map and _map_sharp, among all the output files you can download from the refinement
job in cryoSPARC.<br>
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</p>
<p>At this resolution I would not expect to see noise speckles in the unfiltered/unsharpened map (at a contour level adequate to visualize the protein region).<br>
</p>
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<p>I hope this helps,<br>
</p>
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<p>Guillaume</p>
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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 Francesca GUBELLINI <fgubelli@pasteur.fr><br>
<b>Sent:</b> Friday, March 15, 2024 4:03:56 PM<br>
<b>To:</b> Takanori Nakane<br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Post-processing of dataset from carbon-coated grids: noise pb</font>
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<div>Dear all,
<div class=""><br class="">
<div class="">thank you for you inputs, we wanted to know if there is a special way /trick for carbon-coated grids, since we are not used to work on these grids.
<div class=""><br class="">
<div class="">To answer some of your questions:</div>
<div class=""><br class="">
</div>
<div class="">- We are working on a large complex (600kDa, about 250A) <b class="">
which is very flexible </b></div>
<div class=""><br class="">
</div>
<div class="">- we used commercial-carbon coated Qfoil R1.2/1.3, so carbon is 2nm</div>
<div class=""><br class="">
</div>
<div class="">- Processing was done using Cryosparc and a large box size extraction (980px Fourier-cropped to 320px).</div>
<div class=""><br class="">
</div>
<div class="">- We collected 20,000 images on a Titan Krios operating at 300kV (Flacon 4i) yielding about 200k ptcls after 2D classifications and 92,400 ptcls in the 'final' map</div>
<div class=""><br class="">
</div>
<div class="">When we mentioned 'noise in the map' is that at 9.5A we see some features that we do not think are coming from the protein. Indeed it is possible that we are experiencing over-sharpening (pls see FSC plot attached).</div>
<div class=""><br class="">
</div>
<div class="">Can you suggest us some parameters that can be important to increase the map quality (in Cryosparc)?</div>
<div class=""><br class="">
</div>
<div class="">Thanks again,</div>
<div class=""><br class="">
</div>
<div class="">Francesca and Sophia</div>
<div class=""><br class="">
</div>
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<div class="">---------------------------------</div>
<div class="">Francesca Gubellini, PhD<br class="">
Institut Pasteur<br class="">
Unité de Microbiologie Structurale<br class="">
Bat. Metchnikoff<br class="">
25 rue du Dr Roux<br class="">
75015 Paris<br class="">
France<br class="">
Tel: +33 1 40 61 39 84<br class="">
----------------------------------<br class="">
<br class="">
</div>
<div><br class="">
<blockquote type="cite" class="">
<div class="">On 15 Mar 2024, at 00:11, Takanori Nakane <<a href="mailto:tnakane.protein@osaka-u.ac.jp" class="">tnakane.protein@osaka-u.ac.jp</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class=""><span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Hi,</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">>
we have a question concerning post-processing of pctls from</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">>
carbon-coated grids.</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">></span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">>
We see a lot of noise in our map.</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">We
need more information!</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">What
do you mean by "a lot of noise"?</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Which
software did you use?</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Was
the map bad from the beginning, or did it become</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">bad
after a certain step of processing?</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Does
the FSC curve look reasonable?</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Isn't
the map over-sharpened?</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">If
there are huge variations in the local resolution,</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">one
number might not be adequate.</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">In
general, there is nothing special about carbon-coated grids.</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Best
regards,</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">Takanori
Nakane</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class="">On
3/15/24 01:17, Francesca GUBELLINI wrote:</span><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<blockquote type="cite" style="font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
Dear EM community,<br class="">
we have a question concerning post-processing of pctls from carbon-coated grids.<br class="">
We see a lot of noise in our map.<br class="">
Anyone has specific tips on how to process this kind of data?<br class="">
Thanks a lot!<br class="">
Francesca and Sophia<br class="">
---------------------------------<br class="">
Francesca Gubellini, PhD<br class="">
Institut Pasteur<br class="">
Unité de Microbiologie Structurale<br class="">
Bat. Metchnikoff<br class="">
25 rue du Dr Roux<br class="">
75015 Paris<br class="">
France<br class="">
Tel: +33 1 40 61 39 84<br class="">
----------------------------------<br class="">
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<a href="mailto:3dem@ncmir.ucsd.edu" style="font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px;" class="">3dem@ncmir.ucsd.edu</a><br style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;" class="">
<a href="https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!JFdNOqOXpB6UZW0!vgzwcKmrfMqW1hesFx1r2A4HlEekj6yVqtkx5yD5wdx-_wpPYeGfrt1k5aK5kALHzI71dBcxl1XBX08dZxThEfUQnyyx4wQM7C0$" style="font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px;" class="">https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!JFdNOqOXpB6UZW0!vgzwcKmrfMqW1hesFx1r2A4HlEekj6yVqtkx5yD5wdx-_wpPYeGfrt1k5aK5kALHzI71dBcxl1XBX08dZxThEfUQnyyx4wQM7C0$</a><span style="caret-color: rgb(0, 0, 0); font-family: ArialMT; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;" class=""></span></div>
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När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: <a href="https://urldefense.com/v3/__http://www.uu.se/om-uu/dataskydd-personuppgifter/__;!!Mih3wA!FxTpoA_HHYSkO1TZtrRZQf6Xrzacrsk3qqprsPdw5g1bFgMnmeAUgA7sjbWeLsZBGYVEaAsdEpvYRlXOh6kl5XmMAtiERT2A$">http://www.uu.se/om-uu/dataskydd-personuppgifter/</a>
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E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: <a href="https://urldefense.com/v3/__http://www.uu.se/en/about-uu/data-protection-policy__;!!Mih3wA!FxTpoA_HHYSkO1TZtrRZQf6Xrzacrsk3qqprsPdw5g1bFgMnmeAUgA7sjbWeLsZBGYVEaAsdEpvYRlXOh6kl5XmMAmtsIaWr$">http://www.uu.se/en/about-uu/data-protection-policy</a>
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