<html>
<head>
<meta http-equiv="content-type" content="text/html; charset=utf-8">
</head>
<body text="#000000" bgcolor="#FFFFFF">
<p><br>
</p>
<div class="moz-forward-container"><!--[if gte mso 9]><xml>
<o:OfficeDocumentSettings>
<o:AllowPNG/>
</o:OfficeDocumentSettings>
</xml><![endif]-->
<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"><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"><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">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>
</body>
</html>