Measurement worth not very much if simultaneously error of measurement is not considered. 10 counts intensity on the top of 10000 counts background is close to nothing. 10 counts on the top of 1 count background is an excellent intensity. Simultaneously with diffraction several types of X-ray scattering events occurs such as: Scattering from the instrument parts can be regarded as one of the sources of systematic errors (apertures, beam-stops, peenholes) the should be eliminated or just disregarded Scattering from the air - more or less similar to latter but can be conveniently subtracted and will not produce much systematic errors Scattering from the water in crystal, sometime complicated by powder diffraction appearance Scattering from phonons of the crystalline lattice (TDS) These two are legitimate contributors to the negative net intensity which can be (see Andrew Lesley and Bernard Santasiero comments) negative. What is "true value of Inet" I do not know. I know that Inet can be properly measured according to accepted protocols and 101 of counting statistics in the nuclear physics. And a contribution of the negative intensity processed by F&W or similar (maybe OZ and WM and others) into the quality of the structure and electron density map is MEASURABLE. It is documented already very long time ago: [10] Hirshfeld, F.L.; Rabinowich, D. Treating Weak Reflexions in Least-Squares Calculations. Acta Crystallogr. 1973, A29, 510–513. [11] Arnberg, L.; Hovmo¨ller, S.; Westman, S. On the Significance of ‘Non-Significant’ Reflexions. Acta Crystallogr. 1979, A35, 497–499 However these papers are related to small molecule structures. I personally do not think that protein crystals from point of view of diffraction physics are different from that from crystals of small molecules.
Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Jun 20, 2013, at 20:59 , Douglas Theobald <dtheob...@brandeis.edu> wrote: > On Jun 20, 2013, at 1:47 PM, Felix Frolow <mbfro...@post.tau.ac.il> wrote: > >> Intensity is subtraction: Inet=Iobs - Ibackground. Iobs and Ibackground >> can not be negative. Inet CAN be negative if background is higher than >> Iobs. > > Just to reiterate, we know that the true value of Inet cannot be negative. > Hence, the equation you quote is invalid and illogical --- it has no physical > or statistical justification (except as an approximation for large Iobs and > low Iback, when ironically background correction is unnecessary). That > equation does not account for random statistical fluctuations (e.g., simple > Poisson counting statistics of shot noise). > > >> We do not know how to model background scattering modulated my molecular >> transform and mechanical motion of the molecule, >> I recall we have called it TDS - thermal diffuse scattering. Many years ago >> Boaz Shaanan and JH were fascinated by it. >> If we would know how deal with TDS, we would go to much nicer structures >> some of us like and for sure to much lower >> R factors all of us love excluding maybe referees who will claim over >> refinement :-\ >> Dr Felix Frolow >> Professor of Structural Biology and Biotechnology, >> Department of Molecular Microbiology and Biotechnology >> Tel Aviv University 69978, Israel >> >> Acta Crystallographica F, co-editor >> >> e-mail: mbfro...@post.tau.ac.il >> Tel: ++972-3640-8723 >> Fax: ++972-3640-9407 >> Cellular: 0547 459 608 >> >> On Jun 20, 2013, at 20:07 , Douglas Theobald <dtheob...@brandeis.edu> wrote: >> >>> How can there be nothing "wrong" with something that is unphysical? >>> Intensities cannot be negative. How could you measure a negative number of >>> photons? You can only have a Gaussian distribution around I=0 if you are >>> using an incorrect, unphysical statistical model. As I understand it, the >>> physics predicts that intensities from diffraction should be gamma >>> distributed (i.e., the square of a Gaussian variate), which makes sense as >>> the gamma distribution assigns probability 0 to negative values. >>> >>> >>> On Jun 20, 2013, at 1:00 PM, Bernard D Santarsiero <b...@uic.edu> wrote: >>> >>>> There's absolutely nothing "wrong" with negative intensities. They are >>>> measurements of intensities that are near zero, and some will be negative, >>>> and others positive. The distribution around I=0 can still be Gaussian, >>>> and you have true esd's. With F's you used a derived esd since they can't >>>> be formally generated from the sigma's on I, and are very much >>>> undetermined for small intensities and small F's. >>>> >>>> Small molecule crystallographers routinely refine on F^2 and use all of >>>> the data, even if the F^2's are negative. >>>> >>>> Bernie >>>> >>>> On Jun 20, 2013, at 11:49 AM, Douglas Theobald wrote: >>>> >>>>> Seems to me that the negative Is should be dealt with early on, in the >>>>> integration step. Why exactly do integration programs report negative Is >>>>> to begin with? >>>>> >>>>> >>>>> On Jun 20, 2013, at 12:45 PM, Dom Bellini <dom.bell...@diamond.ac.uk> >>>>> wrote: >>>>> >>>>>> Wouldnt be possible to take advantage of negative Is to >>>>>> extrapolate/estimate the decay of scattering background (kind of Wilson >>>>>> plot of background scattering) to flat out the background and push all >>>>>> the Is to positive values? >>>>>> >>>>>> More of a question rather than a suggestion ... >>>>>> >>>>>> D >>>>>> >>>>>> >>>>>> >>>>>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >>>>>> Ian Tickle >>>>>> Sent: 20 June 2013 17:34 >>>>>> To: ccp4bb >>>>>> Subject: Re: [ccp4bb] ctruncate bug? >>>>>> >>>>>> Yes higher R factors is the usual reason people don't like I-based >>>>>> refinement! >>>>>> >>>>>> Anyway, refining against Is doesn't solve the problem, it only postpones >>>>>> it: you still need the Fs for maps! (though errors in Fs may be less >>>>>> critical then). >>>>>> -- Ian >>>>>> >>>>>> On 20 June 2013 17:20, Dale Tronrud >>>>>> <det...@uoxray.uoregon.edu<mailto:det...@uoxray.uoregon.edu>> wrote: >>>>>> If you are refining against F's you have to find some way to avoid >>>>>> calculating the square root of a negative number. That is why people >>>>>> have historically rejected negative I's and why Truncate and cTruncate >>>>>> were invented. >>>>>> >>>>>> When refining against I, the calculation of (Iobs - Icalc)^2 couldn't >>>>>> care less if Iobs happens to be negative. >>>>>> >>>>>> As for why people still refine against F... When I was distributing >>>>>> a refinement package it could refine against I but no one wanted to do >>>>>> that. The "R values" ended up higher, but they were looking at R >>>>>> values calculated from F's. Of course the F based R values are lower >>>>>> when you refine against F's, that means nothing. >>>>>> >>>>>> If we could get the PDB to report both the F and I based R values >>>>>> for all models maybe we could get a start toward moving to intensity >>>>>> refinement. >>>>>> >>>>>> Dale Tronrud >>>>>> >>>>>> >>>>>> On 06/20/2013 09:06 AM, Douglas Theobald wrote: >>>>>> Just trying to understand the basic issues here. How could refining >>>>>> directly against intensities solve the fundamental problem of negative >>>>>> intensity values? >>>>>> >>>>>> >>>>>> On Jun 20, 2013, at 11:34 AM, Bernhard Rupp >>>>>> <hofkristall...@gmail.com<mailto:hofkristall...@gmail.com>> wrote: >>>>>> As a maybe better alternative, we should (once again) consider to refine >>>>>> against intensities (and I guess George Sheldrick would agree here). >>>>>> >>>>>> I have a simple question - what exactly, short of some sort of historic >>>>>> inertia (or memory lapse), is the reason NOT to refine against >>>>>> intensities? >>>>>> >>>>>> Best, BR >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> -- >>>>>> >>>>>> This e-mail and any attachments may contain confidential, copyright and >>>>>> or privileged material, and are for the use of the intended addressee >>>>>> only. 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