[3dem] Don't blame your thermometer...

Marin van Heel marin.vanheel at googlemail.com
Wed Aug 28 07:28:37 PDT 2013


"Sorry officer I did not know I was speeding, I don't have a gold 
standard speedometer!"

Steven:

(Trolling?  Wikipedia: In Internet slang, a troll (/?tro?l/, /?tr?l/) is 
a person who sows discord on the Internet by starting arguments or 
upsetting people...)

It is not just Sjors in his recent paper who refers to bad processing by 
adding adjectives to the FSC; but it is certainly the clearest case. I 
do notice as a referee that a new generation of scientists are confused 
by the matter and some use it more as a buzz-word without any 
understanding of the underlying issues. Therefore I do think the 
semantics are important here.

Few people take the trouble to look at the older papers on reference bias:
  [
Boekema EJ, Berden JA, van Heel M: *Structure of mitochondrial F1-ATPase 
studied by electron microscopy and image processing*. /Bioch. //Biophys. 
Acta/*851* (1986) 353-360 ]
and to the general risk of introducing artificial similarities between 
the two 3D structures being compared (note that this is the FSC paper):
  [ Harauz G, van Heel M : *Exact filters for general geometry three 
dimensional reconstruction*, /Optik/ *73* (1986) 146-156 ].

Or a bit more modern/recent (only 10 years old) on the importance of 
independent 3D refinements:
[Yang S, Yu X, Galkin VE, Egelman EH: *Issues of resolution and 
polymorphism in single-particle reconstruction.* /J. of Structural 
Biology/ *144* (2003) 162--171. ]
[ Stewart A, Grigorieff N: *Noise bias in the refinement of structures 
derived from single particles.* /Ultramicroscopy/ *102* (2004) 67-84 ].

Cheers,


On 28-Aug-13 9:54 AM, Steven Ludtke wrote:
>> Ahh, but does your thermostat have a wet bulb or a dry bulb 
>> thermometer  ;^)
>>
>> First I thought, "No, what an obvious trolling attempt", then I 
>> decided there may be some actual confusion, so perhaps we should 
>> resolve the semantics. So I will succumb to the urge to reply this 
>> one time (and almost certainly regret doing so later).
>>
>> True, a "gold standard FSC" means "gold standard refinement, followed 
>> by a normal FSC", since that is too long to say, people have started 
>> saying "gold standard FSC".  Neither does cryoEM refer to microscopy 
>> with cold electrons.
>>
>> For anyone who is confused, "gold standard refinement" simply refers 
>> to the process of splitting ones data in half at the very beginning, 
>> and performing two completely independent refinements (with 
>> independent starting models). It is, of course, not the only way of 
>> preventing resolution exaggeration due to model/noise bias, but 
>> (barring the use of other artifact inducers like hard spherical 
>> masks), it is certainly one robust technique for doing so. If you 
>> follow this technique honestly, then you can be confident that your 
>> resolution is not over-estimated. However, most people apply some 
>> sort of mask to reduce noise on the final structure before FSC 
>> computation (otherwise small box sizes lead to better resolutions but 
>> worse structures). This can raise concerns about exaggeration again, 
>> if masking isn't done with an appropriate bias-minimizing mask.
>>
>> * Scheres, S. H. & Chen, S. (2012) Prevention of overfitting in 
>> cryo-EM structure determination. Nat. Methods. 9, 853-854.
>>
>> * Murray, S. C., Flanagan, J., Popova, O. B., Chiu, W., Ludtke, S. J. 
>> & Serysheva, I. I. (2013) Validation of Cryo-EM Structure of IP3R1 
>> Channel. Structure. 21, 1-10. PMC3696195
>>
>> So, if you have any doubts, or are using some other method which you 
>> believe avoids bias, Richard's recent suggestion to randomize the 
>> phases in your raw data, then rerefine to prove that your algorithm 
>> doesn't claim to have achieved resolutions beyond this point can be 
>> used to really test your process.
>>
>> * Chen, S., McMullan, G., Faruqi, A. R., Murshudov, G. N., Short, J. 
>> M., Scheres, S. H. & Henderson, R. (2013) High-resolution noise 
>> substitution to measure overfitting and validate resolution in 3D 
>> structure determination by single particle electron 
>> cryomicroscopy. Ultramicroscopy.
>>
>>

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