Dear Ivan,
On Thu, 1 Aug 2019 22:10:36 -0400, Ivan Shabalin
<[email protected]> 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/
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