Dear Nika,
The question is about a Fo-Fc map and the replies have been focused on such
maps.
However, I question why you are bothering with Fo-Fc maps.
The ligand was soaked into the crystal. There is probably an isomorphous apo
data set available.
A [Fo(ligand complex) - Fo(apo)]*exp(alpha_apo,calc) map should be consulted in
preference to various Fo-Fc maps corrected for phase bias.
It would be even better to substitute in the experimental phases for the apo
structure if
they are available.
Fo-Fo maps may be nosier than Fo-Fc maps. but they are more reliable when you
are
trying to decide if a ligand is present.
I recommend reading the following book chapter:
@incollection{rould2003isomorphous,
title={Isomorphous difference methods},
author={Rould, Mark A and Carter Jr, Charles W},
booktitle={Methods in enzymology},
volume={374},
pages={145--163},
year={2003},
publisher={Elsevier}
}
I second Robbie's "bloody obvious" rule.
Sounds like you have an occupancy issue.
If you collected data from multiple crystals of the complex
as is the standard practice these days,
process and check each one. There can be large differences
in ligand occupancy between crystals from different drops.
Best regards,
Blaine
Blaine Mooers, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology
College of Medicine
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center (BRC) Rm. 466
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419
________________________________________
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of John R Helliwell
[jrhelliw...@gmail.com]
Sent: Wednesday, November 25, 2020 4:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] phenix.refine with ligand with ambiguous
electron density
Hello Robbie,
Yes exactly, I agree. I thought that was what the poster faced: density with
insufficient detail and not extending sufficiently for the whole ligand.
To make the discussion thread more focussed a screenshot or two would assist us.
Greetings,
John
Emeritus Professor John R Helliwell DSc
On 25 Nov 2020, at 09:03, Robbie Joosten <robbie_joos...@hotmail.com> wrote:
I’m with Dale on this, the scientifically prudent thing is to set the rules and
then play by them. Not to change the rules as you go. Of course, in a teaching
environment where you know the correct answer, it is good to be educational and
learn how to dig a bit more.
However, in a scientific setting this digging is not to come to a strong
conclusion, but only to see if you should pursue the project and do additional
experiments (e.g. longer soaks or using a higher ligand concentration). In this
case the topic starter has poor density and fitting the ligand and refining
gives negative difference density. Surely that is not enough evidence to reject
the null hypothesis “the ligand is not bound”. In other words, there is no
strong evidence that the ligand is bound. Perhaps you can look at the occupancy
, but that is probably as far as you should go. The polder map is useful to get
rid of the effect of the solvent mask blurring actual ligand density. But after
fitting the ligand you shouldn’t need the polder map. Blurring and sharpening
is something to make sense of the density shape to better fit your ligand, not
to conclude whether or not you ligand is there.
On a whole, for ligands we should try to stick to the so-called “bloody
obvious” test: if the density is not bloody obvious, your ligand is not there.
At least not all the time.
Cheers,
Robbie
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Jon Cooper
Sent: Wednesday, November 25, 2020 05:20
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density
Hello Dale, the statistical rigour you describe is, of course, excellent, but
in a learning environment, if someone gets a negative result, you have to go
into overdrive to check that everything has been done correctly, since there is
a fair chance that human error is the cause. It may be a terrible practice, but
it would seem to be an important part of the process? Even as a relative
newcomer to the field (well, since the mid-80's ;-) I have seen many people
getting nothing in their initial difference maps, even if the ligand is there.
Frequently it was just the contour level being too high and, depending on how
far back you go, the solution varied from showing someone how to roll the mouse
wheel in Coot to having the map recontoured at a computer centre 200 miles away
and posted back on a magnetic tape, which took about 10 days - a timescale on
which some people just gave up and did something else! I can't help thinking it
would be a shame to robotically accept every negative result at face value, not
least if you're doing something important like curing a pandemic. However, back
to the original question which I think was whether polder map coefficients
could be used as refinement targets and I think the answer to that one is
probably 'no', at least in the X-ray field ;-)
Best wishes, Jon Cooper
-------- Original Message --------
On 24 Nov 2020, 16:02, Dale Tronrud <
de...@daletronrud.com<mailto:de...@daletronrud.com>> wrote:
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<mailto: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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