Hi,
To me, this sounds like a very dangerous way to use this tool decide
if a ligand has bound. I would be very reluctant to modify my map with
a range of arbitrary parameters until it looked like what I wanted to
see. The sharpening and blurring of this tool is not guided or limited
by theory or data.
As you describe it, your choice of map is driven by its agreement
with your ligand, and the proper way to make this decision is the other
way around.
The original poster has the problem that their density does not have
the appearance they desire. They have chosen to run around trying to
find some way to modify the map to get a variant that does. This is a
terrible practice, since the final choice of map is being made in a
fashion that is dominated by bias.
I have no idea what sort of "structural characteristics" have
convinced this poster of the presence of their ligand despite the
absence of clear electron density. What other evidence does a
diffraction pattern give? The map is your best and only source of
information about your structure that you can get from the diffraction
pattern. (Mass spec and other experimental techniques could, of course,
be applied.)
I think we, as a community, could learn a few things from the
vaccine trial studies that are so much in the news now. In a modern
clinical trial, to avoid bias in the interpretation of the results, all
of the statistical procedures are decided upon BEFORE the study is even
began. This protocol is written down and peer reviewed at the start.
Then the study is performed and the protocol is followed exactly. If
the results don't pass the test, the treatment is not supported. There
is no hunting around, after the fact, for a "better" statistical measure
until one is found that "works".
This way of handling data analysis in clinical trials was adopted
after the hard lesson was learned that many trails could be reproduced,
their results were not.
I would recommend that you decide what sort of map you think is the
best at showing features of your active site, based on the resolution of
your data set and other qualities of your project, before you calculate
your first Fourier transform. If you think a Polder map is the bee's
knees then calculate a Polder map and live with it. If you are
convinced of the value of a FEM, or a Buster map, or a SA omit map, or
whatever, calculate that map instead and live with it.
If you have to calculate twenty different kinds of maps, with
varying parameters in each, before you find the one that shows the
density for your ligand; it probably didn't bind.
Dale Tronrud
On 11/24/2020 5:35 AM, John R Helliwell wrote:
Dear Nika,
A tool I am gaining experience with, but for a challenge like you
describe, may help:-
In Coot>Calculate you see “Blurring/Sharpening tool”. You are
presented with a choice of electron density map (here you would select
your Fo-Fc). There is then a slider tool, to the left and to the right,
and you can see the impact of negative or positive B factor on your map.
Blurring, slide right, may assist your density continuity versus
Sharpening, slide left, which may assist the detail of your map. The
logic of the tool is that your diffraction data, and of the Fo-Fc
differences, can be fine tuned, in or out.
Best wishes,
John
Emeritus Professor John R Helliwell DSc
On 24 Nov 2020, at 11:29, Nika Žibrat <nika.zib...@ki.si> wrote:
Hello,
I have a question about protein-ligand, of which ligand displays an
ambiguous electron density. I am solving a structure of protein with
ligand which was obtained via soaking. Structural characteristics
indicate the ligand is present however the electron density is quite
vague and too small for the size of the whole ligand. I did a Polder
map which showed much larger area of green density. After insertion of
my ligand into the green density in Polder I ran phenix.refine and
there is a lot of red on the spot where the ligand is which was to be
expected. This leaves me wondering how, if even do I incorporate the
polder map data into my refine input.
My question is, how do I continue refining and validating the
structure in this case?
Thank you,
Nika Žibrat
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