<html>
<head>
<meta content="text/html; charset=ISO-8859-1"
http-equiv="Content-Type">
</head>
<body bgcolor="#FFFFFF" text="#000000">
Hi Felix,<br>
<br>
NCS: given state-of-the-art NCS restraints there is (probably) no
clear-cut answer, but there are three ones: "definitely yes",
"definitely no", and "try to find out". Obviously, at low enough
resolution NCS should be always used (say ~2A and lower), simply
because this provides a luxury of additional a priori information to
alleviate the poor data-to-parameters ratio problem. Obviously, at
high enough resolution (~1.5-1.7A or so) NCS should not be used
since the amount of data may be enough to see actual differences
between NCS copies, and using NCS would probably wipe out these
difference (or at least there is such a risk). In the grey area,
~1.7-2.0A, one should try using vs not using NCS to know for sure.<br>
<br>
Also, it may be good to mention that if the NCS groups are selected
perfectly (that for example includes making sure to not apply NCS to
atoms that do not obey NCS) then most likely NCS could be used at
any resolution.<br>
<br>
Rfree: at very high resolution not including 5-10% of the data
probably wouldn't hurt too much (provided that the data complete).
Having free-R may be handy even at subatomic resolution: for
example, to illustrate/prove that using IAS (Interatomic Scatterers
model) or Multipoles actually improves your model and not overfits
the data. Note, when multipolar model is used, it is 32 (or 28 - I
forgot?) refinable parameters per atom, so the data-to-parameters
ratio for a macromolecule may not be that great even at ~0.9-0.7A
resolution! Computing less biased maps is another reason to keep the
free set of reflections: note, the m and D in 2mFo-DFc and mFo-DFc
maps have to be computed using free reflections.<br>
<br>
Pavel<br>
<br>
<br>
On 1/31/12 10:07 AM, Felix Frolow wrote:
<blockquote
cite="mid:4481A9C4-D6A7-4516-946D-4E721B340F3F@post.tau.ac.il"
type="cite">
<div>
<div>
<div><font class="Apple-style-span" size="4">
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">I have a question
of general significance: in what resolution NCS
restrains and Rfree become IRRELEVANT?</div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">Axel Brunger
invented Rfree to save our necks from refining garbage
into the structure distantly looking like protein.</div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">Since than Rfree
was idolized. However, there is a big difference between
structures at 4.1 Angstrom and 1.4 Angstrom. </div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">In small molecule
crystallography we can easily achieve 10 or 20
observations per refined parameter (depends on presence
or absence of inversion center), therefore, no one care
about Rfree in the small molecules community.</div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">In the well
ordered protein structures, the bulk water region is
working against us lowering diffraction strength
contributing to 1/Volume, but it is also on our side
minimizing a volume occupied </div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">by protein
molecules (less atoms, fewer parameters). I have a
structure (not yet published) where for 18000 protein
atoms and about 9000 other atoms (water molecules,
sulfate ions, sugars from cryo-protection etc)</div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">there are
750,000 independent observations. It makes about 28
observations per atom and together with the chemical
observations such as bonds and angles which rarely
differs from their classical values defined by small
structures, if we keep anomalous data properly scaled
and separated (there will be differences in good data
sets that depends on S atoms and some other ions in
solute, or even oxygen atoms) -</div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">we have quite
good ratio of observations per refined parameter.</div>
<div style="margin-top: 0px; margin-right: 0px;
margin-bottom: 0px; margin-left: 0px; font: normal
normal normal 14px/normal Helvetica; ">So my question
is: Do WE and WHAT FOR need to mess with Rfree in
structures of relatively/very high resolutions? </div>
<div><br>
</div>
</font></div>
<div apple-content-edited="true">
Dr Felix Frolow <br>
Professor of Structural Biology and Biotechnology<br>
Department of Molecular Microbiology<br>
and Biotechnology<br>
Tel Aviv University 69978, Israel<br>
<br>
Acta Crystallographica F, co-editor<br>
<br>
e-mail: <a moz-do-not-send="true"
href="mailto:mbfrolow@post.tau.ac.il">mbfrolow@post.tau.ac.il</a><br>
Tel: ++972-3640-8723<br>
Fax: ++972-3640-9407<br>
Cellular: 0547 459 608
</div>
<br>
<div>
<div>On Jan 31, 2012, at 17:35 , Pavel Afonine wrote:</div>
<br class="Apple-interchange-newline">
<blockquote type="cite">
<div>Hi Simon,<br>
<br>
the difference is well illustrated in<br>
<br>
F. Fabiola, A. Korostelev and M. S. Chapman<br>
Acta Cryst. (2006). D62, 227-238<br>
Bias in cross-validated free R factors: mitigation of
the effects of non-crystallographic symmetry<br>
<br>
The question is whether we can reproduce it in the exact
same set of test structures.<br>
<br>
Pavel<br>
<br>
On 1/31/12 1:46 AM, Simon Kolstoe wrote:<br>
<blockquote type="cite">Thanks for the interesting
comments.<br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite">I was just wondering what sort
of "difference" we are expecting to see? Is it just a
case of preventing an artificially lowered Rfree or is
there an expectation to see a difference in the
quality of the electron density?<br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite">Simon<br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite">---------------------------------------------------------------<br>
</blockquote>
<blockquote type="cite">Dr Simon Kolstoe<br>
</blockquote>
<blockquote type="cite">Laboratory for Protein
Crystallography<br>
</blockquote>
<blockquote type="cite">Wolfson Drug Discovery Unit<br>
</blockquote>
<blockquote type="cite">University College London<br>
</blockquote>
<blockquote type="cite">Rowland Hill Street, London NW3
2PF<br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite">Tel: 020 7433 2765<br>
</blockquote>
<blockquote type="cite"><a moz-do-not-send="true"
href="http://www.ucl.ac.uk/%7Ermhasek">http://www.ucl.ac.uk/~rmhasek</a><br>
</blockquote>
<blockquote type="cite">---------------------------------------------------------------<br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite">On 31 Jan 2012, at 08:47, A
Leslie wrote:<br>
</blockquote>
<blockquote type="cite"><br>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">Hi Randy,<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"> I can't
remember if I ever mentioned this to you, but when I
was working on the HepB capsid structure (30 fold
ncs if i remember correctly) I tried using a "thin
shell within a thick shell" method of selecting
Rfree, to avoid the issue that within a thin shell
there are still relationships between those
reflections within the shell and those just outside
it. I forget the details, but I think I used a thin
shell of 1-2 rlps wide for the reflections to be
used for Rfree, but I also excluded from the
refinement reflections within a thick shell 4-5 rlps
wide (the thin shell was in the middle of the thick
shell). Because this excluded so many reflections I
could only have 3 thick/thin shells altogether, so I
chose them at low, middle and highish resolution.<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">The upshot of all this was
that it was no help at all. Almost regardless of,
say, the relative weight I put on the Xray terms, or
anything else I did, I could never get the Rfree to
go up ! The strict NCS restraints were so strong
that the refinement essentially always "behaved".<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">This for me destroyed all my
faith in this thin shell idea !<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">So this is definitely NOT an
example where it worked.<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">I have not sent this to the
bulletin board because my memory of exactly what I
did is a bit hazy, but the message was clear enough.<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">Cheers<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">Andrew<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">On 30 Jan 2012, at 17:06,
Randy Read wrote:<br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">I'd be meaning to contribute
to this debate, and now that I see my name
mentioned...<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">I used to be a very strong
believer in selecting the cross-validation data in
thin shells, when you have NCS. I even had a
recollection (a case of false memory syndrome, it
seems) that we did this for our own case of
20-fold NCS, i.e. four copies of the Shiga-like
toxin B-subunit pentamer cocrystallized with the
Gb3 trisaccharide (Ling et al, 1998).<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">As a believer in thin
shells, I was trying to convince Pavel to put an
option for this in Phenix (like the one in
sftools). He said that he'd never seen any
evidence that it was necessary or made any
difference. So I went back to the Shiga-like
toxin structure and started parallel refinements
from the MR solution, either choosing the
cross-validation data randomly or in thin shells.
And, guess what, I couldn't see any significant
difference in how well the refinement went, even
though I was pretty certain before doing that
experiment that it would make a big difference.
In fact, both refinements went pretty well.<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">So if thin shells aren't
necessary even in an extreme case of NCS, then I
suspect that they're not that useful in the more
usual case of lower-order NCS.<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">In any case, there is a
problem even with the thin shells (which Bart
Hazes pointed out even as he implemented it in
sftools). The theory suggests that reflections
within some distance in reciprocal space of some
reflection or a point related to it by an NCS
rotation should be correlated to the original
reflection. All the points related by rotation
will fall into the same resolution shell but,
since the reciprocal-space distance is related to
the inverse of the diameter of the molecule, the
shell would have to have some thickness, and the
reflections at the edge of the shell would still
be correlated to reflections not in the shell. So
even thin-shell cross-validation doesn't get
around all the theoretical problems.<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">I'd be interested if someone
has an example where it really does make a
difference, but in the meantime it's hard to argue
with Pavel's point of view!<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">Regards,<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">Randy<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">On 30 Jan 2012, at 15:26,
Nathaniel Echols wrote:<br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">On Mon, Jan 30, 2012 at
3:43 AM, Simon Kolstoe<<a
moz-do-not-send="true"
href="mailto:s.kolstoe@ucl.ac.uk">s.kolstoe@ucl.ac.uk</a>>
wrote:<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">I see from a quick
google that it is possible to pick my Rfree's
using thin resolution shells (coz I've got 20
fold NCS), however as I am someone who tries
to avoid the GUI where at all possible,<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">Why? Some things are
simply easier to do in the GUI, or at least more<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">obvious - otherwise we
wouldn't bother writing one.<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">could someone let me
know what the command line way of doing this
is?<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">In phenix.refine, you
probably want something like this (some<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">parameters optional, but
the defaults are probably not what most<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">people expect):<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">xray_data.r_free_flags.generate=True<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">xray_data.r_free_flags.fraction=0.05<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">xray_data.r_free_flags.max_free=None<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">xray_data.r_free_flags.use_dataman_shells=True<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">xray_data.r_free_flags.n_shells=20<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">Randy and Paul claim that
this doesn't help very much with the NCS<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">issue, however.<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite"><br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">-Nat<br>
</blockquote>
</blockquote>
</blockquote>
</blockquote>
</div>
</blockquote>
</div>
</div>
</div>
</blockquote>
<br>
</body>
</html>