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320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">Hi Sjors,</span></p>
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style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">The principles of the Fourier
transform and the
sampling theorem (= stay away from Nyquist) have not changed
over the last 35
years. Yet, I have seen much more FSC misuse recently than I
did in previous decades
(see previous posts). So the 2/3<sup>rd</sup> rule remains an
old principle worth
sticking to!</span></p>
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style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">I agree with you that oversampling
helps! So you
are padding your images with zeros to make then 4 times larger
and then do your
interpolations in Fourier space? An image and its Fourier
transform contain the
same information so - up to a certain level - that is fine
with me. (Up to a certain
level meaning: padding an image with zeroes creates a sharp
cut off at the edge
violating the sampling theorem). </span></p>
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style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"></span><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">But then again, why not just
sample the image
correctly - adhering to the 2/3<sup>rd</sup> rule - instead of
under-sampling your
data? <span style="mso-spacerun:yes"></span><span
style="mso-spacerun:yes"></span>The computations will take
just as
long when you double the sampling rate (= halving the pixel
size) and you would
not be violating the sampling rules. <br>
</span></p>
<p class="MsoNormal"
style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">So “I would encourage you” to do
that! (You may even get away with only a 20% reduction in the
pixel size)</span>
</p>
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style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"></span><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">By the way, for twice oversampling
the FT of a 3D
volume you will need 8 times the volume…! Is that really what
you rely on?</span>
</p>
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style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"></span><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">Have fun,</span>
</p>
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style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
normal;tab-stops:45.8pt 91.6pt 137.4pt 183.2pt 229.0pt 274.8pt
320.6pt 366.4pt 412.2pt 458.0pt 503.8pt 549.6pt 595.4pt 641.2pt
687.0pt 732.8pt"><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB"></span><span
style="font-size:10.0pt;font-family:"Courier
New";mso-fareast-font-family:"Times New Roman";
mso-fareast-language:EN-GB">Marin</span>
</p>
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687.0pt 732.8pt"><span
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mso-fareast-language:EN-GB"> </span></p>
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<![endif]-->=================================================================<br>
<br>
On 12/06/2017 14:23, Sjors Scheres wrote:<br>
</div>
<blockquote type="cite"
cite="mid:38c1f874953c0ff69a856607f058028c.squirrel@mail.mrc-lmb.cam.ac.uk">
<pre wrap="">Hi Marin,
I'm not sure about the tone of this discussion anymore. The 2/3
rule-of-thumb was probably a good idea to check for artefacts when the FSC
was mis-used in many applications in the past.
All interpolations in RELION are in the 2x oversampled Fourier domain.
Tests have shown that these lead to errors in the reconstructions become
negligible compared to many other factors until reaching much closer to
Nyquist than the 2/3 limit you describe. I would encourage you to do these
tests yourself: downscale your data by Fourier-cropping to a pixel size
close to the estimated resolution, and re-refine the structure with the
downsampled images. I'm not sure about IMAGIC, but at least in RELION,
you'll find that the final FSC curves will not deviate until very close to
Nyquist.
Best regards,
Sjors
</pre>
<blockquote type="cite">
<pre wrap="">Hi Sjors,
It is good that you agree that the way I defined the FRC/FSC
normalization - some 35 years ago - makes that filtering does not
primarily affect this function. (A quick & sloppy search in Google
Scholar shows you never cited our original papers on the FRC/FSC so I
was not sure you were familiar with our design considerations)
a) But then you choose to disagree with our 2/3 Nyquist rule (see, for
example: Van Heel et al. (2000) “Towards atomic resolutionâ€; and: van
Heel & Schatz (2005)). Strangely enough you agree and you disagree at
the same time…! Literally you state: “If you've chosen a relatively
low magnification, then you could very well end up with non-zero FSCs
all the way up to Nyquist.†And in the next sentence: “Depending on
which program you use, interpolations will suffer more or less the
closer you go to Nyquistâ€. Indeed, that is exactly the point: if you
under-sample the data you get into problems at high resolutions so
better avoid that problem: stay on the safe side (and simply don't use
"a relatively low magnification")!
b) The FSC oscillating around zero beyond the 2/3rd Nyquist limit is an
internal consistency test showing that you indeed did not under-sample
your data! Since you choose to disagree with the 2/3 Nyquist
under-sampling rule you must necessarily disagree with any such
consistency check.
So how can you disagree/agree with the 2/3 Nyquist rule if it makes
perfect scientific sense? Ah... I see why: there is a looming “conflict
of interest†issue here! If you refine the sampling by,
say, 20% then your computational requirements go up by a factor 2
(~(1/0.8)**3)! (Computational requirements in terms of: CPU, memory
usage, and I/O.) If you do everything "in-core" and on the raw data you
will thus continuously run into resource problems!
In other words, you do agree with the scientific principles we expressed
decades ago, but you nevertheless disagree with the idea overall, since
that suits the way of working you rely on! You accept the fact that
interpolation artifacts intermix with genuine high-resolution results
and that you can no longer tell one from the other. (Referees take heed!)
Cheers ;)
Marin
On 29/05/2017 18:42, Sjors Scheres wrote:
</pre>
<blockquote type="cite">
<pre wrap="">Hi Marin,
I agree with your general point that filtering of the maps does not
matter
for FSC calculation. However, I disagree with your rules a) and b). If
you've chosen a relatively low magnification, then you could very well
end
up with non-zero FSCs all the way up to Nyquist. Depending on which
program you use, interpolations will suffer more or less the closer you
go
to Nyquist, but this does not necessarily keep you from reaching
resolutions beyond 2/3 Nyquist. Regarding your rule c), perhaps I'm
partially to blame, as the solvent mask-corrected FSC that RELION writes
out is capped at zero. I will have a look at the code and try to fix
this
in the next release.
I also agree with Oli that submission of unmasked half-maps plus the
mask
used for FSC calculation would be a good idea. As Alan remarked, you can
already do this (as supplementary maps) at the EMDB. I would encourage
everyone to do this.
Best regards,
Sjors
</pre>
<blockquote type="cite">
<pre wrap="">Sure Oli!
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).
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!
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).
Cheers
Marin
On 28/05/2017 13:38, Oliver Clarke wrote:
</pre>
<blockquote type="cite">
<pre wrap="">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.
That seems worth addressing, and it's fairly straightforward to do so.
Cheers
Oli.
On May 28, 2017, at 1:46 PM, Marin van Heel
<<a class="moz-txt-link-abbreviated" href="mailto:0000057a89ab08a1-dmarc-request@JISCMAIL.AC.UK">0000057a89ab08a1-dmarc-request@JISCMAIL.AC.UK</a>
<a class="moz-txt-link-rfc2396E" href="mailto:0000057a89ab08a1-dmarc-request@JISCMAIL.AC.UK"><mailto:0000057a89ab08a1-dmarc-request@JISCMAIL.AC.UK></a>> wrote:
</pre>
<blockquote type="cite">
<pre wrap="">Dear All,
Much misunderstanding persists on the relatively straightforward
issue of the FSC...
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, */Exact filters for general geometry three
dimensional reconstruction/*, Optik 73 (1986) 146-156."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").
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!This is independent of whether 3D reconstruction #1, or #2, (or
both #1 and #2) is/are filtered or not. This means that filtering of
the maps will NOT affect the FSC!I actually have written a paper
about it (Marin van Heel: */Unveiling ribosomal structures: the final
phases/*. Current Opinions in Structural Biology 10 (2000) 259-264,
ask me for a pdf if you have trouble finding it). Quoting from this
paper: “*/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/.*â€Â
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
interpretationâ€Â
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!
Cheers,
Marin
-------- Forwarded Message --------
Subject: Re: [ccpem] Minimum standards for FSC reporting?
Date: Fri, 26 May 2017 23:08:34 -0400
From: Jillian Chase <a class="moz-txt-link-rfc2396E" href="mailto:jillian.d.chase@GMAIL.COM"><jillian.d.chase@GMAIL.COM></a>
Reply-To: Jillian Chase <a class="moz-txt-link-rfc2396E" href="mailto:jillian.d.chase@GMAIL.COM"><jillian.d.chase@GMAIL.COM></a>
To: <a class="moz-txt-link-abbreviated" href="mailto:CCPEM@JISCMAIL.AC.UK">CCPEM@JISCMAIL.AC.UK</a>
Hi John,
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.
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?
Thanks again,
Jillian
Sent from my iPhone
On May 26, 2017, at 10:22 PM, John Rubinstein
<<a class="moz-txt-link-abbreviated" href="mailto:john.rubinstein@utoronto.ca">john.rubinstein@utoronto.ca</a> <a class="moz-txt-link-rfc2396E" href="mailto:john.rubinstein@utoronto.ca"><mailto:john.rubinstein@utoronto.ca></a>>
wrote:
</pre>
<blockquote type="cite">
<pre wrap="">Hi Jillian,
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?
Best wishes,
John
--
John Rubinstein
Molecular Medicine Program
The Hospital for Sick Children Research Institute
686 Bay Street, Rm. 20-9705
Toronto, ON
Canada
M5G 0A4
Tel: (+001) 416-813-7255
Fax: (+001) 416-813-5022
<a class="moz-txt-link-abbreviated" href="http://www.sickkids.ca/research/rubinstein">www.sickkids.ca/research/rubinstein</a>
<a class="moz-txt-link-rfc2396E" href="http://www.sickkids.ca/research/rubinstein"><http://www.sickkids.ca/research/rubinstein></a>
</pre>
<blockquote type="cite">
<pre wrap="">On May 26, 2017, at 9:03 PM, Jillian Chase
<<a class="moz-txt-link-abbreviated" href="mailto:jillian.d.chase@GMAIL.COM">jillian.d.chase@GMAIL.COM</a> <a class="moz-txt-link-rfc2396E" href="mailto:jillian.d.chase@GMAIL.COM"><mailto:jillian.d.chase@GMAIL.COM></a>>
wrote:
Hi,
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?
Best,
Jillian
Sent from my iPhone
</pre>
<blockquote type="cite">
<pre wrap="">On May 26, 2017, at 8:47 PM, Oliver Clarke <<a class="moz-txt-link-abbreviated" href="mailto:olibclarke@GMAIL.COM">olibclarke@GMAIL.COM</a>
<a class="moz-txt-link-rfc2396E" href="mailto:olibclarke@GMAIL.COM"><mailto:olibclarke@GMAIL.COM></a>> wrote:
Hi all,
Ive seen several high-impact cryoEM structures recently with
"headline" global FSC resolutions that do not seem plausible based
on inspection of the map.
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).
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).
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.
(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!)
Thoughts/debate welcome! :)
Cheers
Oli
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<pre wrap="">--
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<p><br>
</p>
<pre class="moz-signature" cols="72">--
==============================================================
Prof Dr Ir Marin van Heel
Research Professor at:
Laboratório Nacional de Nanotecnologia - LNNano
CNPEM/LNNano, Campinas, Brazil
Brazilian mobile phone +55-19-981809332
(041-19-981809332 TIM)
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