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 resolution”; 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 Nyquist”.  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:
> 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
>
>
>
>
>> 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:
>>> 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
>>> <[log in to unmask]
>>> <mailto:[log in to unmask]>> wrote:
>>>
>>>> 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 <[log in to unmask]>
>>>> Reply-To: 	Jillian Chase <[log in to unmask]>
>>>> To: 	[log in to unmask]
>>>>
>>>>
>>>>
>>>> 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
>>>> <[log in to unmask] <mailto:[log in to unmask]>>
>>>> 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
>>>>>> <[log in to unmask] <mailto:[log in to unmask]>> 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 <[log in to unmask]
>>>>>>> <mailto:[log in to unmask]>> 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
>> --
>> ==============================================================
>>
>>       Prof Dr Ir Marin van Heel
>>
>>       Research Professor at:
>>
>>       Laboratorio Nacional de Nanotecnologia - LNNano
>>       CNPEM/ABTLuS, Campinas, Brazil
>>       Brazilian mobile phone  +55-19-981809332
>>                              (041-19-981809332 TIM)
>>
>> ----------------------------------------------
>>
>>
>


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