There are certainly some problems with REFMAC after an anisotropy correction. The FreeR flags and D corrections are organised in spherical shells, whereas the D aniso volume is elliptical..
As Kay points out the REFMAC map coefficient for a difference map is 0 if there is no measurement so there is no bias in those maps. the 2mFo-DFc maps do set the map coefficient to D Fc if there is no measurement, potentially introducing bias. The thinking is that when a large low resolution term is missing it is better to approximate it to something realistic, rather than set it to 0. For high weak resolution data D Fc will also probably be insignificant and there would be very little gain by including it, and the possibility of introducingbias.. However nowadays the data collection techniques usually provide pretty complete data at low resolution, On Tue, 6 Aug 2019 at 07:44, Kay Diederichs <kay.diederi...@uni-konstanz.de> wrote: > Dear Ivan, > > one thing I forgot to mention: I think the difference maps should not show > model bias due to "fill-in". This is because the term D*Fcalc that is > filled in as a replacement for m*Fobs is just compensated by the term D*Fc > that is subtracted when forming the m*Fobs - D*Fcalc difference > coefficients. > This means that when building the model, you can let yourself be guided > primarily by the difference maps. These will not suffer from model bias due > to fill-in. > > I like the idea of the "shaping the MTZ file" that Robbie suggests, but I > still need to work out the proper sftools commands, like those at > http://staraniso.globalphasing.org/test_set_flags_about.html . > > best, > Kay > > On Mon, 5 Aug 2019 17:44:56 -0400, Ivan Shabalin < > iva...@iwonka.med.virginia.edu> wrote: > > >Dear Kay, > > > >Thanks a lot for your answers! > > > >To my best understanding, REFMAC does not have an option of for > >restoring reflections only in certain resolution shells. But, it should > >not be a problem for datasets with good completeness in low resolution > >shells. Also, refinement against intensities is available only for twin > >refinement. > > > >I will take this as a conclusion for datasets with good low resolution > >completeness: "even if the visual effect of weak reflections on the map > >may be low, the errors in the model coordinates will be less if the weak > >amplitudes are used in refinement" > > > >Thanks! > > > >Ivan > > > > > >With best regards, > >Ivan Shabalin, Ph.D. > >Research Scientist, > >Department of Molecular Physiology and Biological Physics, > >University of Virginia, > >1340 Jefferson Park Avenue, Pinn Hall,Room 4223, > >Charlottesville, VA 22908 > >https://www.linkedin.com/in/shabalinig/ > >https://minorlab.org/person/ivan_s/ > > > >On 8/3/19 04:32, Kay Diederichs wrote: > >> Dear Ivan, > >> > >> On Thu, 1 Aug 2019 22:10:36 -0400, Ivan Shabalin < > iva...@iwonka.med.virginia.edu> wrote: > >> > >>> Dear CCP4BB, > >>> > >>> There seems to be a general consensus for extending data to higher > >>> resolution to include as much meaningful data as possible. "Meaningful" > >>> can be defined in different ways. I heard/read opinions such as 0.5 > >>> CC1/2, 0.3 CC1/2, 0.15 CC1/2, > >> > >> This is numerology - why not 0.33333 or 0.12345? The EM community has > agreed on the "gold standard" of 0.143 for FSC which has a similar > definition as CC1/2 - this value is chosen because the quantity analogous > to CC* is then 0.5 ! > >> > >>> and stepped (paired) refinement. The > >>> latter seems to be one of the most rigorous options according to many > >>> crystallographers. > >>> > >>> Including more data sounds like a good thing, but, it sometimes results > >>> in low completeness in high resolution shells. As far as i understand, > >>> this may result from: > >>> > >>> a) anisotropic diffraction (if a software cuts of resolution in > >>> non-isotropic way) > >>> > >>> b) sub-optimal data collection (e.g. due to limitations of the > >>> instrument, such as minimum detector distance allowed, absence of > kappa, > >>> limits on oscillation range) > >>> > >>> In the commonly referred paper, the completeness is 96% in the highest > >>> shell (Karplus, P. A., & Diederichs, K. (2012). Linking > crystallographic > >>> model and data quality. Science (New York, N.Y.), 336(6084), > 1030–1033.) > >>> In other words, these tests were performed for an almost complete > dataset. > >>> > >>> I used to think that more data is always better, but, as I learned > >>> recently from Clemens Vonrhein, the resulting low completeness may > cause > >>> model bias in the maps. > >> > >> ... due to "fill-in" of missing reflections which is performed by > Refmac and phenix.refine ( > https://www.phenix-online.org/documentation/faqs/refine.html#general); > don't know about other programs. > >> > >>> Indeed, REFMAC by default tries to restore missing reflections, which > >>> are approximated as DFc > >>> ( > https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html > ). > >>> > >>> > >>> We tried using the keyword "mapcalculate free include" and > "mapcalculate > >>> free exclude" for one of our structures (~1.3A, P1), and it did seem to > >>> improve the maps a little - we saw more meaningful features. > >>> > >>> But, I still have several questions: > >>> > >>> 1) Does using "mapcalculate free include" in REFMAC represent a sound > >>> solution to this problem? Does this "no fill-in at all" solution > >>> constitute a significant problem? > >> > >> I think it would be desirable to "fill in" only the reflections missing > at low resolution (e.g. in your case to 3A); that would improve the maps > but decrease the model bias. Not sure if any program supports this. > >> > >>> > >>> 2) Are there any other concerns about using data with low completeness > >>> in highest shells? > >> > >> no. > >> > >> To the contrary, even if the visual effect of weak reflections on the > map may be low, the errors in the model coordinates will be less if the > weak amplitudes (or even better, intensities - once the refinement programs > use these) are used as restraints in refinement. > >> > >>> > >>> 3) STARANISO website suggests a way of handling this problem > >>> (http://staraniso.globalphasing.org/test_set_flags_about.html). But, > >>> would not REFMAC "fill-in" all the reflections for map coefficients > >>> calculation to isotropic completeness anyway? > >>> > >>> 4) What is your personal approach to handling this issue? Is there > >>> completeness value in the last shell that is too low to include it in > >>> Table 1? > >> > >> To me, the rule "the completeness in high-resolution shell is too low > if it is less than XX %" appears not to be based on any appropriate > concept. If there is good information in that resolution shell, it should > be used no matter what the completeness is. Practically, this means that > one has to devote a lot of attention to doing the experiment right, by > choosing an appropriate crystal-detector distance. As always, attempts to > computationally rectify problems that result from a poor experiment have > their own caveats. > >> > >> Thanks for bringing this up! > >> > >> Kay > >> > >>> > >>> Many thanks, > >>> > >>> Ivan > >>> > >>> With best regards, > >>> Ivan Shabalin, Ph.D. > >>> Research Scientist, > >>> Department of Molecular Physiology and Biological Physics, > >>> University of Virginia, > >>> 1340 Jefferson Park Avenue, Pinn Hall,Room 4223, > >>> Charlottesville, VA 22908 > >>> https://www.linkedin.com/in/shabalinig/ > >>> https://minorlab.org/person/ivan_s/ > >>> > >>> > >>> > >>> > >>> > >>> ---- > >>> > >>> > ######################################################################## > >>> > >>> To unsubscribe from the CCP4BB list, click the following link: > >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > >> > >> ######################################################################## > >> > >> To unsubscribe from the CCP4BB list, click the following link: > >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > >> > > > >---- > > > >######################################################################## > > > >To unsubscribe from the CCP4BB list, click the following link: > >https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
