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Sure Oli! <br>
<br>
I fully agree that two maps should always be deposited (for each
3D reconstruction) and that those two maps should be unmasked
(serious errors can be made while masking). <br>
However, the filtering state of the two maps is by itself not so
relevant because of the built-in FSC normalization! That was my
main point!<br>
<br>
Among the many FSC errors that I have seen in the flood of cryo-EM
papers the more serious ones include: a) under-sampling the data
and thus claiming a resolution beyond 2/3 of the Nyquist
frequency; b) the FSC should oscillate around zero beyond 2/3rd
Nyquist whereas in many publications a FSC remains positive up to
the Nyquist frequency, c) in many publication the vertical FSC
axis starts at "0" and goes to "1" so one cannot even verify the
oscillations around the "0" axis. I also don't like using the same
automatically generated 3D mask for the two half volumes. I just
now did a Google image search for "Fourier Shell Correlation" and
below is the result. I have no idea whose FSCs I am looking at but
a majority violate at least one of the basic rules (and I am not
even counting the ones using incorrect fixed-valued thresholds
like 0.5 or 0.143).<br>
<br>
Cheers<br>
Marin<br>
<br>
<img src="cid:part1.7A04DAAA.BD5948B7@googlemail.com" alt=""><br>
<br>
<br>
On 28/05/2017 13:38, Oliver Clarke wrote:<br>
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cite="mid:1EB54C19-1AE2-4B03-BEE6-43FC7C0111D6@gmail.com">
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<div>That's all well and good, but without deposition of the
unfiltered half maps and the mask used to calculate the FSC it
is not possible to reproduce the resolution calculations of the
authors, because only one map is deposited, it is sharpened and
low pass filtered, and the mask used for FSC calculation is
often neither deposited nor described. </div>
<div><br>
</div>
<div>That seems worth addressing, and it's fairly straightforward
to do so.</div>
<div><br>
</div>
<div>Cheers</div>
<div>Oli.</div>
<div><br>
On May 28, 2017, at 1:46 PM, Marin van Heel <<a
href="mailto:0000057a89ab08a1-dmarc-request@JISCMAIL.AC.UK"
moz-do-not-send="true">0000057a89ab08a1-dmarc-request@JISCMAIL.AC.UK</a>>
wrote:<br>
<br>
</div>
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<p class="MsoNormal"><span
style="font-size:12.0pt;line-height:107%;font-family:
"Times New Roman",serif">Dear All,</span></p>
<p class="MsoNormal"><span
style="font-size:12.0pt;line-height:107%;font-family:
"Times New Roman",serif">Much misunderstanding
persists on the relatively straightforward issue of the
FSC... </span></p>
<p class="MsoNormal"><span
style="font-size:12.0pt;line-height:107%;font-family:
"Times New Roman",serif">1) In the first
place: please do read the primary literature rather than
relying on second-hand or third-hand references where
errors/misunderstanding have accumulated. The first
mention in the literature of the "Fourier Shell
Correlation" is in "George Harauz and Marin van Heel, <b
style="mso-bidi-font-weight:normal"><i
style="mso-bidi-font-style: normal">Exact filters
for general geometry three dimensional
reconstruction</i></b>, Optik 73 (1986) 146-156."<span
style="mso-spacerun:yes"> </span>The how and why of
the FSC normalization of the amplitudes is explicitly
described in the original paper(s). (You can find more
in Wikipedia: "Fourier Shell Correlation"). </span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;mso-layout-grid-align:none;text-autospace:none"><span
style="font-size: 12.0pt;font-family:"Times New
Roman",serif">2) Now about the consequences of that
normalization: Any filtering that does not zero a
specific spatial frequency will affect the nominator and
the denominator of the FSC equation in exactly the same
way!<span style="mso-spacerun:yes"> </span>This is
independent of whether 3D reconstruction #1, or #2, (or
both #1 and #2) is/are filtered or not. <span
style="mso-spacerun:yes"> </span>This means that
filtering of the maps will NOT affect the FSC!<span
style="mso-spacerun:yes"> </span>I actually have
written a paper about it (Marin van Heel: <b
style="mso-bidi-font-weight:normal"><i
style="mso-bidi-font-style:normal">Unveiling
ribosomal structures: the final phases</i></b>.
Current Opinions in Structural Biology 10 (2000)
259-264, ask me for a pdf if you have trouble finding
it). Quoting from this paper: “<b
style="mso-bidi-font-weight:normal"><i
style="mso-bidi-font-style:normal">The bottom line …
is that there is no wrong way of filtering the data,
as its information content is not normally affected.
The one and only thing one can do wrong is to
interpret the map incorrectly</i>.</b>”</span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;mso-layout-grid-align:none;text-autospace:none"><span
style="font-size: 12.0pt;font-family:"Times New
Roman",serif"> 3) Thus, the fact that you don’t see
certain details in the map for a given level of the FSC
curve probably says more about your representation
choices than about the map. Low-pass filtering a map to
the 0.5 value of the FSC as a way to avoid “over
interpretation” is in general a bad idea. You would
probably be killing (the visibility of) the high-res
info as a self-fulfilling prophecy. On the other hand,
relying entirely on black-box programs that in some
mysterious way boost the visibility of high-res noise
beyond any reasonable FSC value can equally be a bad
idea. Please do keep in mind that the final
interpretation of your map is your own responsibility!</span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;mso-layout-grid-align:none;text-autospace:none"><span
style="font-size: 12.0pt;font-family:"Times New
Roman",serif"> Cheers,</span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;mso-layout-grid-align:none;text-autospace:none"><span
style="font-size: 12.0pt;font-family:"Times New
Roman",serif"> Marin</span></p>
<p class="MsoNormal"><span
style="font-size:12.0pt;line-height:107%;font-family:
"Times New Roman",serif"> </span><br>
</p>
-------- Forwarded Message --------
<table class="moz-email-headers-table" cellspacing="0"
cellpadding="0" border="0">
<tbody>
<tr>
<th nowrap="nowrap" valign="BASELINE" align="RIGHT">Subject:
</th>
<td>Re: [ccpem] Minimum standards for FSC reporting?</td>
</tr>
<tr>
<th nowrap="nowrap" valign="BASELINE" align="RIGHT">Date:
</th>
<td>Fri, 26 May 2017 23:08:34 -0400</td>
</tr>
<tr>
<th nowrap="nowrap" valign="BASELINE" align="RIGHT">From:
</th>
<td>Jillian Chase <a class="moz-txt-link-rfc2396E"
href="mailto:jillian.d.chase@GMAIL.COM"
moz-do-not-send="true"><jillian.d.chase@GMAIL.COM></a></td>
</tr>
<tr>
<th nowrap="nowrap" valign="BASELINE" align="RIGHT">Reply-To:
</th>
<td>Jillian Chase <a class="moz-txt-link-rfc2396E"
href="mailto:jillian.d.chase@GMAIL.COM"
moz-do-not-send="true"><jillian.d.chase@GMAIL.COM></a></td>
</tr>
<tr>
<th nowrap="nowrap" valign="BASELINE" align="RIGHT">To:
</th>
<td><a class="moz-txt-link-abbreviated"
href="mailto:CCPEM@JISCMAIL.AC.UK"
moz-do-not-send="true">CCPEM@JISCMAIL.AC.UK</a></td>
</tr>
</tbody>
</table>
<br>
<br>
<meta http-equiv="content-type" content="text/html;
charset=utf-8">
<div>Hi John,</div>
<div id="AppleMailSignature"><br>
</div>
<div id="AppleMailSignature">Thanks for your reply. It is
possible that I was viewing the unsharpened map. I
imported that map into relion for targeted post-processing
based on threshold values from viewing map in chimera,
resulting in a more reasonable 4A. I'll double check which
I imported. </div>
<div id="AppleMailSignature"><br>
</div>
<div id="AppleMailSignature">Still puzzling though: the
cryosparc map wth post processing in relion shows more
side chain density than what I see with identical particle
set processed in entirety in relion. I've been using a
hybrid of both programs to generate best maps possible.
Has anyone done more quantitative tests using both
programs that may have some input?</div>
<div id="AppleMailSignature"><br>
</div>
<div id="AppleMailSignature">Thanks again,</div>
<div id="AppleMailSignature">Jillian<br>
<br>
Sent from my iPhone</div>
<div><br>
On May 26, 2017, at 10:22 PM, John Rubinstein <<a
href="mailto:john.rubinstein@utoronto.ca"
moz-do-not-send="true">john.rubinstein@utoronto.ca</a>>
wrote:<br>
<br>
</div>
<blockquote type="cite">
<div>
<meta http-equiv="Content-Type" content="text/html;
charset=utf-8">
Hi Jillian,
<div class=""><br class="">
</div>
<div class="">Recently in our group one cryoSPARC users
was accidentally downloading structures from the
experiments overview page rather than getting the
sharpened final maps from the experiment details page.
The maps from the experiments overview page can be
selected for further processing but are not sharpened
and will look worse than expected for their
resolution. Is it possible you’ve been looking at the
unsharpened maps?</div>
<div class=""><br class="">
</div>
<div class="">Best wishes,</div>
<div class="">John</div>
<div class=""><br class="">
<div class="">
<div style="color: rgb(0, 0, 0); letter-spacing:
normal; orphans: auto; text-align: start;
text-indent: 0px; text-transform: none;
white-space: normal; widows: auto; word-spacing:
0px; -webkit-text-stroke-width: 0px; word-wrap:
break-word; -webkit-nbsp-mode: space;
-webkit-line-break: after-white-space;" class="">
<div class="">
<div class="">-- </div>
<div class="">John Rubinstein</div>
<div class="">Molecular Medicine Program</div>
<div class="">The Hospital for Sick Children
Research Institute</div>
<div class="">686 Bay Street, Rm. 20-9705</div>
<div class="">Toronto, ON</div>
<div class="">Canada</div>
<div class="">M5G 0A4</div>
<div class="">Tel: (+001) 416-813-7255</div>
<div class="">Fax: (+001) 416-813-5022</div>
<div class=""><a
href="http://www.sickkids.ca/research/rubinstein"
class="" moz-do-not-send="true">www.sickkids.ca/research/rubinstein</a></div>
</div>
</div>
</div>
<br class="">
<div>
<blockquote type="cite" class="">
<div class="">On May 26, 2017, at 9:03 PM, Jillian
Chase <<a
href="mailto:jillian.d.chase@GMAIL.COM"
class="" moz-do-not-send="true">jillian.d.chase@GMAIL.COM</a>>
wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div class="">Hi,<br class="">
<br class="">
I've also noticed significantly higher FSC
resolution estimates with cryosparc vs relion,
which do not seem realistic upon inspection.
(IE: a 4A relion postprocessed map looks much
different than a 4A cryosparc map). Has anyone
noticed as well? How are you handling?<br
class="">
<br class="">
Best,<br class="">
Jillian<br class="">
<br class="">
Sent from my iPhone<br class="">
<br class="">
<blockquote type="cite" class="">On May 26,
2017, at 8:47 PM, Oliver Clarke <<a
href="mailto:olibclarke@GMAIL.COM"
class="" moz-do-not-send="true">olibclarke@GMAIL.COM</a>>
wrote:<br class="">
<br class="">
Hi all,<br class="">
<br class="">
Ive seen several high-impact cryoEM
structures recently with "headline" global
FSC resolutions that do not seem plausible
based on inspection of the map.<br class="">
<br class="">
In each case, the resolution was based on
results out of relion_postprocess, but no
details were given about mask calculation or
the volume of the mask compared to the
model, and only the final map was deposited,
not the half maps (so checking workings was
not possible).<br class="">
<br class="">
I think that at a bare minimum, reporting
either the volume of the mask compared to
the volume of the map at the suggested
contour level, or simply displaying an
overlay of the mask on the model, should be
mandatory (as should deposition of
unfiltered half maps to facilitate
recalculation of the FSC). <br class="">
<br class="">
Without knowledge of the mask, the FSC is
meaningless, particularly if the author has
chosen to use relion_postprocess as a "black
box", and has chosen to automatically
generate a mask based on an initial
threshold without subsequently inspecting
it.<br class="">
<br class="">
(There have also been a couple of structures
using the pymol 'carve' option in extremely
misleading ways without disclosing its use
or the map contour level, but that is a rant
for another day!)<br class="">
<br class="">
Thoughts/debate welcome! :)<br class="">
<br class="">
Cheers<br class="">
<br class="">
Oli<br class="">
</blockquote>
</div>
</div>
</blockquote>
</div>
<br class="">
</div>
</div>
</blockquote>
</div>
</div>
</blockquote>
</blockquote>
<pre class="moz-signature" cols="72">--
==============================================================
Prof Dr Ir Marin van Heel
Research Professor at:
Laboratório Nacional de Nanotecnologia - LNNano
CNPEM/ABTLuS, Campinas, Brazil
Brazilian mobile phone +55-19-981809332
(041-19-981809332 TIM)
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