<html>
<head>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8">
</head>
<body text="#000000" bgcolor="#FFFFFF">
<div class="moz-cite-prefix"><br>
<br>
Dear Teige,<br>
<br>
You state "... whether you use a 1/2 bit FSC criterion ... or a
0.143 FSC criterion justified by Peter, Tony and Richard... " as
it there is a equal choice.<br>
On the contrary: we show the "0.143 FSC criterion" to be WRONG ,
because it is based on flawed ("sloppy") statistics! Unfortunately
this applies to almost all resolution criteria in use in cryo-EM
(see our BioRxiv paper). In science you have to simply accept you
made a mistake, lick you wounds, and move on. The discussion
during the last two weeks was primarily about the consequences of
under-sampling the data a closely related but equally widely
misunderstood topic. <br>
<br>
Chiara, the FSC is a 1D curve and its behavior below the 1/2 bit
threshold may be an important thing to look at
(<a class="moz-txt-link-freetext" href="http://journals.iucr.org/m/issues/2017/05/00/kf5002/index.html">http://journals.iucr.org/m/issues/2017/05/00/kf5002/index.html</a>).<br>
<br>
Have fun,<br>
<br>
Marin<br>
<br>
PS: this slide is from my Wiley lecture in April 2017 and which I
also used in my presentation in Stockholm on the 9th of Dec 2017
at the cryo-EM symposium during the Nobel 2017 festivities. <br>
<br>
<img src="cid:part1.B817011E.2966EDBA@googlemail.com" alt=""><br>
<br>
<br>
<br>
On 08/09/2018 07:26, Teige Matthews-Palmer wrote:<br>
</div>
<blockquote type="cite"
cite="mid:7BE52DD8-B598-40CF-B401-199374A187F7@crick.ac.uk">
<meta http-equiv="Content-Type" content="text/html; charset=utf-8">
Dear Chiara,
<div class=""><br class="">
</div>
<div class="">My (non-expert) take on it is that, whether you use
a 1/2 bit FSC criterion (asymptotic to 0.1716), advocated by
Marin: <a
href="https://www.biorxiv.org/content/biorxiv/early/2017/11/24/224402.full.pdf"
class="" moz-do-not-send="true">https://www.biorxiv.org/content/biorxiv/early/2017/11/24/224402.full.pdf</a> or
a 0.143 FSC criterion justified by Peter, Tony and Richard in
this appendix: <a
href="https://www.sciencedirect.com/science/article/pii/S0022283603010222?via=ihub#APP1"
class="" moz-do-not-send="true">https://www.sciencedirect.com/science/article/pii/S0022283603010222?via%3Dihub#APP1</a> ,
these fall-off thresholds require some assumptions to be met.
E.g. FSC is between two independent half-maps; FSC vs resolution
curve is a ‘nice shape’ - a threshold shouldn’t replace looking
at the curve, which should be high before a single fall-off
without big oscillations.</div>
<div class="">There is a debate going on about whether reporting a
resolution beyond particular fractions of Nyquist is valid, but
your negative stain reconstruction will be too far from Nyquist
for you to worry about that.</div>
<div class="">Whereas a half-map FSC for high-res cryo is trying
to determine a threshold where correlated signal can be
interpreted as scattering by the molecules, negative stain is
giving you a lot of signal from scattering by variable stain
envelopes. </div>
<div class="">I think so long as the FSC curve is not a weird
shape or oscillating, you should be able to use the same FSC
criterion as cryo, for low-res. I’d love to hear more
experienced views on this.</div>
<div class=""><br class="">
</div>
<div class="">Another quality measure could be to look at your
map’s anisotropy. Relion for example automatically produces a 3D
histogram of assigned particle angles, and Dmitry Lyumkis has
shared his group’s program for measuring anisotropy with FSC: <a
href="https://mail.ncmir.ucsd.edu/pipermail/3dem/2017-September/005210.html"
class="" moz-do-not-send="true">https://mail.ncmir.ucsd.edu/pipermail/3dem/2017-September/005210.html</a></div>
<div class=""><br class="">
</div>
<div class="">Finally, you might want to know if the shape of your
3D map at low-res is correct, which is definitely not a given.
You can collect tilt-pairs (or do tomography): <a
href="https://www.sciencedirect.com/science/article/pii/S0959440X15000925"
class="" moz-do-not-send="true">https://www.sciencedirect.com/science/article/pii/S0959440X15000925</a> but
I think there have been other validation methods suggested that
don’t require extra data, but I can’t find the paper. T_T Does
anyone know?</div>
<div class=""><br class="">
</div>
<div class="">All the best,</div>
<div class="">Teige</div>
<div class=""><br class="">
</div>
<div class=""><br class="">
<div>
<blockquote type="cite" class="">
<div class="">On 8 Sep 2018, at 01:00, Dmitry Lyumkis <<a
href="mailto:dlyumkis@SALK.EDU" class=""
moz-do-not-send="true">dlyumkis@SALK.EDU</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div dir="auto" class="">
<div class="">Weekend FSC spar 2.0?</div>
<div class=""><br class="">
</div>
<div class=""><br class="">
On Sep 7, 2018, at 11:34 AM, Chiara Rapisarda <<a
href="mailto:c.rapisarda@IECB.U-BORDEAUX.FR"
class="" moz-do-not-send="true">c.rapisarda@IECB.U-BORDEAUX.FR</a>>
wrote:<br class="">
<br class="">
</div>
<blockquote type="cite" class="">
<div class="">
<div style="font-family: arial, helvetica,
sans-serif; font-size: 12pt;" class="">
<div class="">Dear community members,<br class="">
</div>
<div class=""><br data-mce-bogus="1" class="">
</div>
<div class="">I would like to know what is the
state of the art for reporting negative stain
reconstruction resolution.
<br data-mce-bogus="1" class="">
</div>
<div class="">Is it best to not include it or to
use the 0.5 FSC cut off. I definitely don't want
to use the 0.143 cut-off, but I thought that it
is important to give an idea of the quality of
the reconstruciton by using some form of
quantitative measure.<br data-mce-bogus="1"
class="">
</div>
<div class=""><br data-mce-bogus="1" class="">
</div>
<div class="">Is there an agreement on what to do?
<br data-mce-bogus="1" class="">
</div>
<div class="">Thank you for any feedback I will
receive.<br class="">
</div>
<div class=""><br data-mce-bogus="1" class="">
</div>
<div data-marker="__SIG_PRE__" class="">Chiara
Rapisarda <br class="">
<br class="">
Post doc <br class="">
Université de Bordeaux, CNRS, INSERM <br
class="">
<a href="mailto:c.rapisarda@iecb.u-bordeaux.fr"
class="" moz-do-not-send="true">c.rapisarda@iecb.u-bordeaux.fr</a>
<br class="">
Tel. +33 (5) 4000 3617 <br class="">
<br class="">
Institut européen de chimie et biologie <br
class="">
2, rue Robert Escarpit <br class="">
33607 Pessac, France <br class="">
<a href="http://www.iecb.u-bordeaux.fr/"
class="" moz-do-not-send="true">www.iecb.u-bordeaux.fr</a>
<br class="">
<br class="">
</div>
</div>
<br class="">
</div>
</blockquote>
</div>
</div>
</blockquote>
</div>
</div>
</blockquote>
<br>
<pre class="moz-signature" cols="72">--
==============================================================
Prof Dr Ir Marin van Heel
Laboratório Nacional de Nanotecnologia - LNNano
CNPEM/LNNano, Campinas, Brazil
email: marin.vanheel(A_T)gmail.com
marin.vanheel(A_T)lnnano.cnpem.br
and: mvh.office(A_T)gmail.com
--------------------------------------------------
I receive many emails per day and, although I try,
there is no guarantee that I will actually read each incoming email. </pre>
</body>
</html>