Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Dirk Kostrewa]

A way to achieve a feeling for the significance of the different map 
features that you are looking at in your current project, is the old 
control technique to omit both a well ordered part of the model 
(residue, alpha-helical turn, part of a beta-strand, ...) and a less 
well ordered part (residue, loop, end of visible secondary structure, 
...). Then you run refinement until convergence and calculate the 
different maps that you want to try. If you now look at the appearance 
of these maps at different contour levels for both the well and less 
well ordered omitted parts, and compare this with the appearance of the 
same maps at the same contour levels for your questionable part (bound 
ligand?), you can quickly get a feeling, how far you can trust what you 
see in these maps.


I learned this at the beginning of my crystallographic work and still do 
it today if I'm in doubt.


Cheers,

Dirk.

On 03.12.20 10:20, Robert Nicholls wrote:

Hi Dale,

You're absolutely right - the multiple hypothesis testing problem is 
one that is often not considered, let alone properly accounted for. 
Whilst this can be accounted for by appropriate adjustment of 
significance levels when a known number of explicit hypotheses are 
tested (and when estimated sigmas are appropriate and reliable...), 
this is extremely difficult in the present context when we passively 
conduct a large number of quick map evaluations subjectively by eye. 
Objective guidelines in such a case, which don't essentially boil down 
to an automated procedure, or unduly inhibit the process in other 
ways, would be valuable. I don't think there's a clear answer to this 
today, although raising awareness of such issues is very prudent. 
Indeed, there is an outstanding need for additional approaches for 
cross-validation, and perhaps re-evaluation of policies 
regarding provision of evidence of the reproducibility of 
crystallographic models. You're correct to say that, ultimately, there 
is (presently) no substitute for education and experience.


Best regards,
Rob


On 3 Dec 2020, at 08:09, Dale Tronrud > wrote:


Hi,

  Dr Nicholls brings up many interesting points, but doesn't touch on 
the major point I had hoped to make in my letter.  Whenever you start 
making multiple tests of your hypothesis you have to evaluate each of 
those tests with a higher standard than you would if you only applied 
one.  If you take a survey of the amount of fat people eat along with 
their history of heart disease you can calculate a correlation and 
find it significant with a p value of 0.05.  If, instead, you perform 
a survey asking for twenty different dietary behaviors and twenty 
health outcomes and find a correlation between eating fat and heart 
disease you need a much higher "signal" to determine its 
significance.  You just made 400 comparisons and a p of 0.05 allows 
20 spurious correlations to appear significant.


  If you are exploring your data set to decide if a compound has 
bound, and your try several different refinement programs and 
calculate several different map types based on the results of those 
refinements, and then adjust the blur of each map, and pick the map 
with the strongest peak in the putative binding site, you have to 
consider the significance of that peak height to be less than if you 
had just calculated one map and got that same height.


  Ignoring this counterintuitive fact has resulted in a huge number 
of studies in many fields to be published that ultimately turned out 
to not be reproducible.  It likely has also resulted in the 
deposition of a lot of "complex" models in the PDB that aren't correct.


  Yes, I am arguing for an ideal, hoping to pull some of you over 
toward my side a bit.  I certainly understand that one has to be 
flexible when solving a difficult problem, but you can't ignore that 
this "flexibility" has significant consequences for understanding the 
results of your work.


  Dr Nicholls' letter brings up a related topic which I'd like to 
explore.  His letter repeatedly mentions the importance of 
"intuition" when interpreting a map.  Yes, the power of human 
intuition, and our inability to replicate it in silico is the reason 
we are still staring at maps in Coot.  Intuition is a remarkable tool 
which, by its nature, is difficult to describe.


  Yet, no one is born with an innate intuition for interpreting 
electron density maps.  Intuition is acquired thru practice. 
 Practice is not simple repetition, however.  You can't become 
proficient in shooting basketball hoops by simply repeatedly throwing 
a basketball on the roof of your garage.  You have to have a proper 
backboard and a hoop.  Now, after repeatedly throwing the ball and 
"feeling" the difference between it going through the hoop and not, 
you will develop the ability to make a basket w/o really thinking 
about it.  You will have developed an intuition for achieving that task.


  There are two caveats.  

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robert Nicholls]

Hi Dale,

You're absolutely right - the multiple hypothesis testing problem is one that 
is often not considered, let alone properly accounted for. Whilst this can be 
accounted for by appropriate adjustment of significance levels when a known 
number of explicit hypotheses are tested (and when estimated sigmas are 
appropriate and reliable...), this is extremely difficult in the present 
context when we passively conduct a large number of quick map evaluations 
subjectively by eye. Objective guidelines in such a case, which don't 
essentially boil down to an automated procedure, or unduly inhibit the process 
in other ways, would be valuable. I don't think there's a clear answer to this 
today, although raising awareness of such issues is very prudent. Indeed, there 
is an outstanding need for additional approaches for cross-validation, and 
perhaps re-evaluation of policies regarding provision of evidence of the 
reproducibility of crystallographic models. You're correct to say that, 
ultimately, there is (presently) no substitute for education and experience.

Best regards,
Rob


> On 3 Dec 2020, at 08:09, Dale Tronrud  wrote:
> 
> Hi,
> 
>   Dr Nicholls brings up many interesting points, but doesn't touch on the 
> major point I had hoped to make in my letter.  Whenever you start making 
> multiple tests of your hypothesis you have to evaluate each of those tests 
> with a higher standard than you would if you only applied one.  If you take a 
> survey of the amount of fat people eat along with their history of heart 
> disease you can calculate a correlation and find it significant with a p 
> value of 0.05.  If, instead, you perform a survey asking for twenty different 
> dietary behaviors and twenty health outcomes and find a correlation between 
> eating fat and heart disease you need a much higher "signal" to determine its 
> significance.  You just made 400 comparisons and a p of 0.05 allows 20 
> spurious correlations to appear significant.
> 
>   If you are exploring your data set to decide if a compound has bound, and 
> your try several different refinement programs and calculate several 
> different map types based on the results of those refinements, and then 
> adjust the blur of each map, and pick the map with the strongest peak in the 
> putative binding site, you have to consider the significance of that peak 
> height to be less than if you had just calculated one map and got that same 
> height.
> 
>   Ignoring this counterintuitive fact has resulted in a huge number of 
> studies in many fields to be published that ultimately turned out to not be 
> reproducible.  It likely has also resulted in the deposition of a lot of 
> "complex" models in the PDB that aren't correct.
> 
>   Yes, I am arguing for an ideal, hoping to pull some of you over toward my 
> side a bit.  I certainly understand that one has to be flexible when solving 
> a difficult problem, but you can't ignore that this "flexibility" has 
> significant consequences for understanding the results of your work.
> 
>   Dr Nicholls' letter brings up a related topic which I'd like to explore.  
> His letter repeatedly mentions the importance of "intuition" when 
> interpreting a map.  Yes, the power of human intuition, and our inability to 
> replicate it in silico is the reason we are still staring at maps in Coot.  
> Intuition is a remarkable tool which, by its nature, is difficult to describe.
> 
>   Yet, no one is born with an innate intuition for interpreting electron 
> density maps.  Intuition is acquired thru practice.  Practice is not simple 
> repetition, however.  You can't become proficient in shooting basketball 
> hoops by simply repeatedly throwing a basketball on the roof of your garage.  
> You have to have a proper backboard and a hoop.  Now, after repeatedly 
> throwing the ball and "feeling" the difference between it going through the 
> hoop and not, you will develop the ability to make a basket w/o really 
> thinking about it.  You will have developed an intuition for achieving that 
> task.
> 
>   There are two caveats.  First, you have to actually watch the ball go 
> through the hoop.  If you close your eyes right after your throw you will 
> never develop a useful skill.  It is the feedback from the success or failure 
> of each attempt that makes it practice.  Second, no matter how much time you 
> spend shooting baskets, you will never get better at dribbling the ball.  
> Good practice allows you to develop intuition, but only intuition about that 
> task.
> 
>   Let's say you are working on a project, but having difficulty interpreting 
> your map at some critical location.  You ask around and learn of some spiffy 
> new map calculation and you want to try it.  While you certainly can 
> calculate the map, you have no intuition on how to interpret it.  You have 
> not practiced with that type of map.
> 
>   It may look similar to the maps you've looked at before, but that 
> similarity can be a 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Dale Tronrud]

Hi,

   Dr Nicholls brings up many interesting points, but doesn't touch on 
the major point I had hoped to make in my letter.  Whenever you start 
making multiple tests of your hypothesis you have to evaluate each of 
those tests with a higher standard than you would if you only applied 
one.  If you take a survey of the amount of fat people eat along with 
their history of heart disease you can calculate a correlation and find 
it significant with a p value of 0.05.  If, instead, you perform a 
survey asking for twenty different dietary behaviors and twenty health 
outcomes and find a correlation between eating fat and heart disease you 
need a much higher "signal" to determine its significance.  You just 
made 400 comparisons and a p of 0.05 allows 20 spurious correlations to 
appear significant.


   If you are exploring your data set to decide if a compound has 
bound, and your try several different refinement programs and calculate 
several different map types based on the results of those refinements, 
and then adjust the blur of each map, and pick the map with the 
strongest peak in the putative binding site, you have to consider the 
significance of that peak height to be less than if you had just 
calculated one map and got that same height.


   Ignoring this counterintuitive fact has resulted in a huge number of 
studies in many fields to be published that ultimately turned out to not 
be reproducible.  It likely has also resulted in the deposition of a lot 
of "complex" models in the PDB that aren't correct.


   Yes, I am arguing for an ideal, hoping to pull some of you over 
toward my side a bit.  I certainly understand that one has to be 
flexible when solving a difficult problem, but you can't ignore that 
this "flexibility" has significant consequences for understanding the 
results of your work.


   Dr Nicholls' letter brings up a related topic which I'd like to 
explore.  His letter repeatedly mentions the importance of "intuition" 
when interpreting a map.  Yes, the power of human intuition, and our 
inability to replicate it in silico is the reason we are still staring 
at maps in Coot.  Intuition is a remarkable tool which, by its nature, 
is difficult to describe.


   Yet, no one is born with an innate intuition for interpreting 
electron density maps.  Intuition is acquired thru practice.  Practice 
is not simple repetition, however.  You can't become proficient in 
shooting basketball hoops by simply repeatedly throwing a basketball on 
the roof of your garage.  You have to have a proper backboard and a 
hoop.  Now, after repeatedly throwing the ball and "feeling" the 
difference between it going through the hoop and not, you will develop 
the ability to make a basket w/o really thinking about it.  You will 
have developed an intuition for achieving that task.


   There are two caveats.  First, you have to actually watch the ball 
go through the hoop.  If you close your eyes right after your throw you 
will never develop a useful skill.  It is the feedback from the success 
or failure of each attempt that makes it practice.  Second, no matter 
how much time you spend shooting baskets, you will never get better at 
dribbling the ball.  Good practice allows you to develop intuition, but 
only intuition about that task.


   Let's say you are working on a project, but having difficulty 
interpreting your map at some critical location.  You ask around and 
learn of some spiffy new map calculation and you want to try it.  While 
you certainly can calculate the map, you have no intuition on how to 
interpret it.  You have not practiced with that type of map.


   It may look similar to the maps you've looked at before, but that 
similarity can be a trap.  By now a large number of us here on the BB 
have had the experience of looking at a high resolution electrostatic 
potential (ESP) map and "feeling" that something is wrong with it.  The 
carbonyl oxygen bumps are too small and the acid groups are oddly weak. 
 Wow, those magnesium ions really stand out -- Maybe they're potassium 
instead?  No, there is nothing wrong with the ESP map.  The fault is 
with our intuition which was based on many, many hours of looking at ED 
maps.  To interpret ESP maps you have to practice with a bunch of ESP 
maps first.


   You cannot develop intuition for the spiffy map calculated from your 
project's data since you don't know its correct interpretation -- It 
cannot give you feedback.  Before you calculate this map for your data 
you should calculate versions for many other *completed* projects and 
get a "feel" for what that kind of map shows under different 
circumstances.  Practice, practice, practice, then you will be ready to 
return to your little mystery and be able to apply your, newly acquired, 
intuition.


   Yes, I try new refinement programs - But first I run refinement with 
them on familiar proteins. Yes, I try new styles of map calculations - 
But first I calculate those maps for 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robert Nicholls]

Dear all,
 
I feel the need to respond following last week’s critique of the use of Coot’s 
map blurring tool for providing diagnostic insight and aiding ligand 
identification…

> On 24 Nov 2020, at 16:02, Dale Tronrud  > wrote:
> 
> 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.

I disagree with this, subject to the important qualification that care is 
needed with interpretation. Blurring isn't a crime - it merely involves 
adjusting the weighting given to lower versus higher resolution reflections, 
and thus allows relaxation of the choice of high-resolution limit, and 
facilitates local investigation of regions that exhibit a poor signal-to-noise 
ratio. This is particularly pertinent to liganded compounds, which are 
typically present with sub-unitary occupancies.
 
Coot's blurring merely involves convolution of the whole map with an isotropic 
3D Gaussian, with a parameter (B-factor) to control the standard deviation of 
the Gaussian. This corresponds to reweighting the structure factors in order to 
give higher weight to lower-resolution reflections. This approach is guided by 
a very simple theory: higher resolution structure factors (SFs) are typically 
noisier, with a worse signal-to-noise ratio than lower resolution SFs (due to 
increased errors in both observed higher-resolution reflections and calculated 
phases). Consequently, increasing the blurring B-factor reduces the effect of 
the noisier higher-resolution SFs. This results in a map that should be more 
reliable, but at the expense of reduced structural detail due to artificially 
reducing the effective resolution.
 
It should be noted that this does assume that lower resolution reflections are 
more reliable than higher resolution ones. So, good low-resolution data quality 
and completeness is important.
 
Unfortunately, determination of an optimal B-factor parameter is not presently 
automated. Consequently, users are currently expected to trial different values 
in the Coot slider tool in order to maximise information and gain, for want of 
a better word, intuition. Furthermore, due to the spatially heterogeneous 
nature of atomic positional uncertainty in macromolecular complexes, it can be 
that different B-factor parameters are of optimal usefulness in different local 
regions of the map that exhibit different signal-to-noise ratios. Such issues 
are on-going areas of research.
 
The main problem is that interpretation is subjective. In difficult cases, it 
is necessary to obtain as much information and insight as possible in order to 
gain a good intuition. If you can't see a ligand in the "standard" maps, but 
you can see evidence for a ligand in blurred density (or difference density) 
maps of the various types, then it means that careful exploration of those 
avenues is required. Any "evidence" from viewing such maps and map types should 
serve to guide intuition, and should be digested along with all other available 
information. Such complementary maps should be seen as diagnostics to gain 
intuition, rather than something that can be used as an unequivocal argument 
for ligand binding.
 
Ultimately, the presence of significant density in a blurred map means that 
there is something substantial present. Or in a blurred difference density that 
there is something missing from the current model. This could be a missing 
ligand, or it could be a mismodelled region of the macromolecule, or it could 
be mismodelled solvent (in which case re-evaluating any solvent mask may be 
worthwhile). Ultimately it is down to the practitioner to explore all potential 
explanations for any such behaviour, in order to maximise intuition and 
convince themselves of the crystal's structural composition.
 
In some cases the presence of density in a blurred map might be sufficient to 
convince the practitioner that it is worth pursing investigation of binding. 
This may take various forms: hypothesising an approximate pose for the ligand; 
the nature of interactions in the structural environment of the macromolecule; 
re-evaluation after modelling and refinement; or simply stating that there may 
be evidence of binding. In many cases, the latter is the appropriate action, 
and, as Robbie quite rightly pointed out: "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".

> On 24 Nov 2020, at 16:02, Dale Tronrud  > wrote:
> [...] 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 

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Clemens Vonrhein]

Dear all,

coming back to those map questions again:

On Wed, Nov 25, 2020 at 12:56:16PM +, Mooers, Blaine H.M. (HSC) wrote:
> 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. 

Generally I would agree, but with a slight caveat: the accurate
scaling of those two sets of Fo can become tricky. If the Fo(apo) was
collected in a very different way (different beamline, different
detector, different protocol, different processing package, re-using a
PDB deposition from 10 years ago etc) compared to Fo(soak), things can
become difficult: the required scaling of noisy measurements to extract
the very small ligand signal has to be very accurate.

So when doing high- or low-throughput ligand screening data
collection, maybe we shoulld always collect another APO dataset during
teh same beamline shift? Of course, this then start to play the same
role as the "ground-state" map in PanDDA (if I understood this
correctly) ... but a very isomorphous (in terms of experiment,
hardware, processing) experimental APO dataset seems like a good idea
nevertheless.

> I second Robbie's "bloody obvious" rule. 

Ditto.

I like to start with the null hypothesis that the experiment (soaking
a crystal with a compound solution) has not worked (ligand has not
bound). That seems a better "bias" than the other way round ("I soaked
therefore it is there.").

Our ligand-detection maps approach three questions in turn - each
depending on a positive outcome of the previous:

  (1) Has something bound (=> detection of residual difference density
  under the null hypothesis)?

  (2) Where has it bound (=> detection of what we call "interesting
  regions")?

  (3) How has it bound (=> removal or potentially wrong dummy
  water/atoms to result in shape description of binding excluding
  bulk-solvent)

So instead of zooming in on our favourite binding site (which fits so
nicely that beautiful theory I have) and immediately dialing map
levels down or playing with B-factor sharpening, it forces us to take
several steps back from any potential bias I think. Note that map
levels and B-factor sharpening/blurring are very useful tools that can
become crucial in the correct interpretation of compound density, but
they tend to mainly modify shape and levels while easily pushing the
initial question ("Has this small compound bound?") into the background.

Anyway, just another minor thoughts ...

Cheers

Clemens

 https://www.globalphasing.com/buster/wiki/index.cgi?DifferenceFourierMaps#iso
 https://www.globalphasing.com/buster/wiki/index.cgi?LigandDetectionModes



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robert Nicholls]

Dear all,

Coming back to map blurring...

> On 24 Nov 2020, at 16:41, John R Helliwell  > wrote:
> 
> [...] Coot’s blurring and sharpening tool is tethered directly to one’s 
> measured diffraction data. 
> [...] But, I have just rechecked the Coot manual and see no reference.


The use of Coot's blurring tool is briefly recommended in the context of ligand 
binding analysis on page 12 of this publication: 
https://doi.org/10.1107/S2059798316020143 
. This is so useful in cryo-EM that 
the technique of simultaneously viewing multiple maps with different levels of 
blurring/sharpening as a visual confirmatory aid has become routine 
(https://doi.org/10.1107/S2059798318007313 
). More generally, map blurring can 
be useful for exploring evidence for the presence of structure in the crystal 
that is currently missing from the model in both MX and cryo-EM.

Regards,
Rob


> On 27 Nov 2020, at 08:41, Clemens Vonrhein  wrote:
> 
> Dear Nika,
> 
> as a possible alternative or second opinion, you could have a look at
> the ligand-detection modes [1] in BUSTER too - see e.g.
> 
>  https://www.globalphasing.com/buster/wiki/index.cgi?LigandDetectionModes
> 
> It's very similar to the Phenix Polder maps - so might not tell you
> anything different, but looking at results from different
> implementations can be useful as a check.
> 
> Anyway, one of the main problems handling partially occupied ligands
> (and let's ignore the possibility of partial disorder for
> "simplicity") is that you might have a mixture of three models within
> your binding site: (1) compound, (2) waters (at ordered positions when
> the ligand is not bound) and (3) bulk solvent (at disordered positions
> when the ligand is not bound).
> 
> What can be useful is to include at least the alternative ordered
> water model (from a well refined APO model?) and then refine the
> (grouped) occupancies:
> 
>  OCC(compound) + OCC(waters) = 1.0
> 
> This way you give the refinement the option to chose between two
> alternative interpretations [2]. To avoid any bias in that refinement
> procedure, you could start from two extremes: once with
> OCC(compound)=0.9 and once with 0.1. If the OCC(compound) refines to a
> small value (say <0.2 or such) in both cases, it is possibly not really
> there.
> 
> Cheers
> 
> Clemens
> 
> [1] Vonrhein, C. and Bricogne, G., 2005. Automated structure
>refinement for high‐throughput ligand detection with
>BUSTER‐TNT. Acta Crystallogr. Sect. A, 61, p.c248.
> [2] 
> http://www.globalphasing.com/pipermail/buster-discuss/2015-August/000255.html
> 
> On Tue, Nov 24, 2020 at 11:28:42AM +, Nika Žibrat 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
>> 
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
>> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing 
>> list hosted by www.jiscmail.ac.uk, terms & conditions are available at 
>> https://www.jiscmail.ac.uk/policyandsecurity/
> 
> -- 
> 
> *--
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> * Global Phasing Ltd., Sheraton House, Castle Park 
> * Cambridge CB3 0AX, UK   www.globalphasing.com
> *--
> 
> 
> 
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> 
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This 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Clemens Vonrhein]

Dear Nika,

as a possible alternative or second opinion, you could have a look at
the ligand-detection modes [1] in BUSTER too - see e.g.

  https://www.globalphasing.com/buster/wiki/index.cgi?LigandDetectionModes

It's very similar to the Phenix Polder maps - so might not tell you
anything different, but looking at results from different
implementations can be useful as a check.

Anyway, one of the main problems handling partially occupied ligands
(and let's ignore the possibility of partial disorder for
"simplicity") is that you might have a mixture of three models within
your binding site: (1) compound, (2) waters (at ordered positions when
the ligand is not bound) and (3) bulk solvent (at disordered positions
when the ligand is not bound).

What can be useful is to include at least the alternative ordered
water model (from a well refined APO model?) and then refine the
(grouped) occupancies:

  OCC(compound) + OCC(waters) = 1.0

This way you give the refinement the option to chose between two
alternative interpretations [2]. To avoid any bias in that refinement
procedure, you could start from two extremes: once with
OCC(compound)=0.9 and once with 0.1. If the OCC(compound) refines to a
small value (say <0.2 or such) in both cases, it is possibly not really
there.

Cheers

Clemens

[1] Vonrhein, C. and Bricogne, G., 2005. Automated structure
refinement for high‐throughput ligand detection with
BUSTER‐TNT. Acta Crystallogr. Sect. A, 61, p.c248.
[2] 
http://www.globalphasing.com/pipermail/buster-discuss/2015-August/000255.html

On Tue, Nov 24, 2020 at 11:28:42AM +, Nika Žibrat 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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*--
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
* Global Phasing Ltd., Sheraton House, Castle Park 
* Cambridge CB3 0AX, UK   www.globalphasing.com
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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Dorothee Liebschner]

Hi Nika,

- As Herman wrote, you should use common sense to interpret a polder map:
if the ligand is not there, this kind of map may show only bulk solvent. So
the appearance of some density in the area of the ligand does not "prove"
that the ligand is there. Also, map interpretation should be done while
keeping in mind data quality, data resolution and the state of the model
(how far along you are in refinement). Ligand density may not be clear yet
if the model is fresh out of MR, but it may become better once the model is
finalized. At high resolution, negative density may appear if the occupancy
is too high and it may disappear if you refine it (of course, don't let it
refine to unreasonably low occupancy...).

- There is no need to refine against a polder map. If the ligand is placed
in the model, the bulk solvent mask is calculated accordingly, so
refinement is aware that there is no bulk solvent in this area.

I wrote the polder tool, so if you want, I can have a look at the maps.
Send me the model with the refined ligand + data (+ cif restraints if
applicable). If you send files, be mindful to do so off-list (reply only to
me).

Best wishes,

Dorothee


On Tue, Nov 24, 2020 at 3:29 AM Nika Žibrat  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
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
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Lawrence Berkeley National Laboratory
1 Cyclotron Road, M/S 33R0345
Berkeley, CA 94720
Tel: (510) 486-5709
Fax: (510) 486-5909
Web: https://phenix-online.org



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Bernhard Rupp]

In reasonably compact format, the discussion topic is summarized in this
CCP4 weekend introduction

https://journals.iucr.org/d/issues/2013/02/00/wd5191/index.html

 

As far as the solvent exclusion self-deception via low occupancy goes, fig 2
here:

https://febs.onlinelibrary.wiley.com/doi/epdf/10./febs.14320

 

Good luck, BR

 

From: CCP4 bulletin board  On Behalf Of Nika Žibrat
Sent: Tuesday, November 24, 2020 03:29
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] phenix.refine with ligand with ambiguous electron density

 

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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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB
 =1 




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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Dale Tronrud]

Dear Jon,

   I don't think we have any disagreement.  I just wanted to emphasize 
that you should have your plan thought out at the start.  You may have 
decided that you will compare your ligands shape with your map using a 
Real Space R Factor.  If you don't like that number the fault isn't in 
the RSR but in your density.


   Of course you could have decided ahead of time to use a correlation 
coefficient.  Or you could have planed to calculate both and defined 
some weighting scheme to use the two in making the decision.  The error 
comes in if you decision tree depends on how well the analysis justifies 
your desired outcome.


   Like your test set, how you make your decisions should be kept 
isolated from your actual analysis.  The power of the human mind to bend 
its choices toward a rewarding outcome, even unconsciously, is enormous.


   I was trying to stay away from the particulars of what that decision 
tree would look like.  That topic has been discussed many times on the 
BB.  I certainly agree with my good friend Blaine Moors that the Fo-Fo 
map is the gold standard for deciding if something bound and gives a map 
that is unbiased by modeling.  In addition, Fo-Fo maps between the 
crystals of varying occupancy, even very small changes in occupancy, are 
surprisingly informative.  They tend to be highly isomorphorus and 
provide direct information for deconvoluting multiple conformations 
which is vital in partial occupancy binding.


Dale Tronrud

P.S. Changing the contour level does not change the map. That is simply 
a representation issue due to the difficulty of presenting all the 
information in a map.  Sharpening or blurring a map makes a new map, and 
since the sharpening factor is a continuous number that dial wheel 
creates an infinite number of different maps.  If your only means of 
selecting which one is "best" is how well the map fits your ligand, that 
map can't be used to justify your interpretation.


This could be made rigorous by, for example, deciding on the factor by 
looking at the quality of the map in some uncontroversial region -- If 
you decide on the means of choosing that region ahead of time.


On 11/24/2020 8:20 PM, Jon Cooper wrote:
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> 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

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Mooers, Blaine H.M. (HSC)]

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  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  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 refineme

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[John R Helliwell]

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  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  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> 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 t

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Robbie Joosten]

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  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 trai

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Jon Cooper]

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 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  wrote:
>>>
>>> 
>>>
>>> Hello,
>>>
>>>
>>> I have a 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[John R Helliwell]

Hello Dale,
Well, warming to your theme, I start with a trust in Coot before a new project.
Secondly, Coot’s blurring and sharpening tool is tethered directly to one’s 
measured diffraction data. 
Thirdly, scrutinising it at a sigma level above 5, Coot’s default, is certainly 
not the same as delving into the lower levels of a map’s sigma (and truly of 
noise).
But, I have just rechecked the Coot manual and see no reference. There are 
several google hits but mainly to cryoEM maps. 
It does seem a free lunch though..
Greetings,
John 


Emeritus Professor John R Helliwell DSc




> On 24 Nov 2020, at 16:02, Dale Tronrud  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  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 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Dale Tronrud]

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  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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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Jon Cooper]

Hello, I was wondering if you are refining the ligand occupancy. Eleanor 
mentioned resolution which is important here. If it's good enough, occupancy 
refinement of the ligand or the fragment will clean the map up, assuming the 
occupancy is much less than one. Sorry, if I'm just saying the obvious...

Best wishes, Jon Cooper
 Original Message 
On 24 Nov 2020, 11:28, Nika Žibrat 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
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Andrew Mesecar]

Hi Nika,

A couple of other things you could try to improve maps to add to what the
others have suggested.  One, I am assuming that you are refining the
occupancy of the ligand, but if not, that should reduce the negative
density?  2.  Since you mention soaking the ligand, do you have a good
X-ray data set and refined structure of the unliganded protein in the same
space group and about the same resolution?  If so, you could run an
Fo(ligand)-Fo(unliganded) isomorphorous difference map calculation using
the phases of the unliganded structure for a map calculation.  This may
help (or hurt) too.

Best wishes
Andy Mesecar

On Tue, Nov 24, 2020 at 6:30 AM Nika Žibrat  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
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
*Andrew D. Me**secar*
Head, Department of Biochemistry
Walther Professor of Cancer Structural Biology
Deputy Director, Purdue Center for Cancer Research
E-Mail: amese...@purdue.edu
_
*Department of Biochemistry Contact Information:*
175 S. University Street
W. Lafayette, IN 47907-2063
765-494-1607
--
*Research Lab Contact Information:*
Hockmeyer Hall of Structural Biology
Room 311
240 S. Martin Jischke Drive
West Lafayette, IN 47907-1971
765-494-1924
_



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[John R Helliwell]

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  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
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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