[3dem] [ccpem] Minimum standards for FSC reporting?

Sun May 28 04:46:12 PDT 2017

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/.*”

  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!

  Cheers,

  Marin

-------- Forwarded Message --------
Subject: 	Re: [ccpem] Minimum standards for FSC reporting?
Date: 	Fri, 26 May 2017 23:08:34 -0400
From: 	Jillian Chase <jillian.d.chase at GMAIL.COM>
Reply-To: 	Jillian Chase <jillian.d.chase at GMAIL.COM>
To: 	CCPEM at JISCMAIL.AC.UK

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 
<john.rubinstein at utoronto.ca <mailto:john.rubinstein at utoronto.ca>> wrote:

> 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
> www.sickkids.ca/research/rubinstein 
> <http://www.sickkids.ca/research/rubinstein>
>
>> On May 26, 2017, at 9:03 PM, Jillian Chase <jillian.d.chase at GMAIL.COM 
>> <mailto:jillian.d.chase at GMAIL.COM>> 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
>>
>>> On May 26, 2017, at 8:47 PM, Oliver Clarke <olibclarke at GMAIL.COM 
>>> <mailto:olibclarke at GMAIL.COM>> 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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