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Hi Maia,<br>
<br>
the atom parameters, such as occupancy, B-factor and even position are
interdependent in some sense. That is, if you have somewhat incorrect
occupancy, that B-factor refinement may compensate for it; if you
misplaced an atom the refinement of its occupancy or/and B-factor will
compensate for this. Note in all the above cases the 2mFo-DFc and
mFo-DFc maps will appear almost identical, as well as R-factors.<br>
<br>
So, I think your goal of finding a "true" occupancy is hardly
achievable. <br>
<br>
Although, I think you can approach it by doing very many refinements
(say, several hundreds) (where you refine occupancies, B-factors and
coordinates) each refinement starting with different occupancy and
B-factor values, and make sure that each refinement converges. Then
select a subset of refined structures with similar and low R-factors
(discard those cases where refinement got stuck for whatever reason and
R-factors are higher) (and probably similar looking 2mFo-DFc and
mFo-DFc maps in the region of interest). Then see where the refined
occupancies and B-factors are clustering, and the averaged values will
probably give you an approximate values for occupancy and B. I did not
try this myself but always wanted to.<br>
<br>
If you have a structure consisting of 9 carbons and one gold atom, then
I would expect that the "second digit" in gold's occupancy would
matter. However, if we speak about dozen of ligand atoms (which are
probably a combination of C,N,O) out of a few thousands of atoms of the
whole structure, then I would not expect the "second digit" to be
visibly important.<br>
<br>
Pavel.<br>
<br>
<br>
On 11/24/09 8:08 PM, chern wrote:
<blockquote cite="mid:45739FA9A7074C69A50CB5412BB18E87@LEO" type="cite">
<title>Re: [phenixbb] occupancy refinement</title>
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<div><font face="Arial" size="2">Thank you Kendall and Pavel for your
responces. </font></div>
<div> </div>
<div><font face="Arial" size="2">I really want</font><font
face="Arial" size="2"> to determine the occupancy of my ligand. I saw
one suggestion to try different refinements with different occupancies
and compare the B-factors.</font></div>
<div><font face="Arial" size="2">The occupancy with a B-factor
that is at the level with the average protein B-factors, is a "true"
occupancy. </font></div>
<div><font face="Arial" size="2"> I also noticed the dependence of
the ligand occupancy on the initial occupancy. I saw the difference of
10 to 15%, that is why I am wondering if the second digit after the
decimal point makes any sence.</font></div>
<div> </div>
<div><font face="Arial" size="2">Maia</font></div>
<div> </div>
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style="border-left: 2px solid rgb(0, 0, 0); padding-right: 0px; padding-left: 5px; margin-left: 5px; margin-right: 0px;">
<div
style="font-family: arial; font-style: normal; font-variant: normal; font-weight: normal; font-size: 10pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">-----
Original Message ----- </div>
<div
style="background: rgb(228, 228, 228) none repeat scroll 0%; -moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial; font-family: arial; font-style: normal; font-variant: normal; font-weight: normal; font-size: 10pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"><b>From:</b>
<a moz-do-not-send="true" title="knettles@scripps.edu"
href="mailto:knettles@scripps.edu">Kendall Nettles</a> </div>
<div
style="font-family: arial; font-style: normal; font-variant: normal; font-weight: normal; font-size: 10pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"><b>To:</b>
<a moz-do-not-send="true" title="phenixbb@phenix-online.org"
href="mailto:phenixbb@phenix-online.org">PHENIX user mailing list</a> </div>
<div
style="font-family: arial; font-style: normal; font-variant: normal; font-weight: normal; font-size: 10pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"><b>Sent:</b>
Tuesday, November 24, 2009 8:22 PM</div>
<div
style="font-family: arial; font-style: normal; font-variant: normal; font-weight: normal; font-size: 10pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"><b>Subject:</b>
Re: [phenixbb] occupancy refinement</div>
<div><br>
</div>
<font face="Verdana, Helvetica, Arial"><span
style="font-size: 12px;">Hi Maia, <br>
I think the criteria for occupancy refinement of ligands is similar to
a decision to add an alt conformation for an amino acid. I don’t
refine occupancy of a ligand unless the difference map indicates that
we have to. Sometimes part of the igand may be conformationally mobile
and show poor density, but I personally don’t think this justifies
occupancy refinement without evidence from the difference map. I agree
with Pavel that you shouldn’t expect much change in overall statistics,
unless the ligand has very low occupancy., or you have a very small
protein. We typically see 0.5-1% difference in R factors from refining
with ligand versus without for nuclear receptor igand binding domains
of about 250 amino acids, and we see very small differences from
occupancy refinement of the ligands.<br>
<br>
Regarding the error, I have noticed differences of 10% percent
occupancy depending on what you set the starting occupancy before
refinement. That is, if the starting occupancy starts at 1, you might
end up with 50%, but if you start it at 0.01, you might get 40%. I
don’t have the expertise to explain why this is, but I also don’t think
it is necessarily important. I think it is more important to convince
yourself that the ligand binds how you think it does. With steroid
receptors, the ligand is usually planer, and tethered by hydrogen bonds
on two ends. That leaves us with with four possible poses, so if in
doubt, we will dock in the ligand in all of the four orientations and
refine. So far, we have had only one of several dozen structures where
the ligand orientation was not obvious after this procedure. I worry
about a letter to the editor suggesting that the electron density for
the ligand doesn’t support the conclusions of the paper, not whether
the occupancy is 40% versus 50%. <br>
<br>
You might also want to consider looking at several maps, such as the
simple or simulated annealing composite omit maps. These can be noisy,
so also try the kicked maps ( <a moz-do-not-send="true"
href="http://www.phenix-online.org/pipermail/phenixbb/2009-September/002573.html%29,">http://www.phenix-online.org/pipermail/phenixbb/2009-September/002573.html),</a>
which I have become a big fan of. <br>
<br>
Regards,<br>
Kendall Nettles<br>
<br>
On 11/24/09 3:07 PM, <a class="moz-txt-link-rfc2396E" href="mailto:chern@ualberta.ca">"chern@ualberta.ca"</a> <a class="moz-txt-link-rfc2396E" href="mailto:chern@ualberta.ca"><chern@ualberta.ca></a>
wrote:<br>
<br>
</span></font>
<blockquote><font face="Verdana, Helvetica, Arial"><span
style="font-size: 12px;">Hi,<br>
I am wondering what is the criteria for occupancy refinement of <br>
ligands. I noticed that R factors change very little, but the ligand <br>
B-factors change significantly . On the other hand, the occupancy is <br>
refined to the second digit after the decimal point. How can I find <br>
out the error for the refined occupancy of ligands?<br>
<br>
Maia<br>
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