Re: [ccp4bb] Occupancy Refinement limitation

[Eleanor Dodson]

Low tech but simple.
Exclude both conformers from the refinement. (set occs to 0.00 so you still
see the coordinates in COOT)
Look at the difference map density and make a rough estimate of occupancy
from peak heights for a "fixed" bit of the conformers..

The relative ratios for different states should show up...
Eleanor

On Wed, 6 Sept 2023 at 11:49,  wrote:

> I would be tempted to try phenix.ensemble_refinement instead.
>
> Look at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949522/
> or
> https://phenix-online.org/phenixwebsite_static/mainsite/files/presentations/ensemble_refinement_burnley_at_al_10DEC2012.pdf
>
> or https://elifesciences.org/articles/00311
>
> Tassos
>
> On 6 Sep 2023, at 12:37, Matt Mcleod  wrote:
>
> Hi,
>
> I should be a bit more specific.
>
> We have many crystal structures to indicate that a loop adopts
> conformation A and conformation B, or - the loop can be disordered where
> the electron density is washed out.  These states are dependent on how we
> perturb the system.
>
> We have a series of data, as a function of osmolyte concentration, that
> adjusts this equilibrium and we want to put a numerical value onto it.
> Resolution is between 1.7 - 2.0 A. This is non-anomalous data but certainly
> would be helpful in the future to include if possible some anomalous
> scatters. Have been using phenix.refine.
>
> So what we have done is modeled both conformation A and B and are refining
> the data in order to get the occupancies (fixing B-factors) regardless of
> if there is electron density present for one of the two conformations since
> in some cases (likely all) there will be a population of both A, B, and
> disordered and the relative true occupancies will move.  I am trying to
> sort out how accurate these occupancy values are as opposed to showing the
> electron density for each conformation fit at some common threshold.  My
> general sense is that if it is modeled but no electron density, there will
> be a non-zero value and vice versa if it is the only conformation present
> it will be less than unity.
>
> I will take a look at the references!  Very much appreciated.
> Matt
>
> On Wed, 6 Sept 2023 at 02:08, Eleanor Dodson <
> 176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Well - occupancy refinement is particularly imprecise, and highly
>> correlated with temperature factors.
>> Also the population for a surface ARG or LYS may well have more than two
>> conformations, whereas some internal residue is better defined.
>> There is also the Q of solvent - dual occupancies will generate dual
>> solvent networks..
>>
>> ..You dont say what resolution your data are. Again at 0.8A you can be
>> confident - at 3A it is at best a guess. So I for one do not take the
>> numbers very seriously - they are a flag only.
>>
>> Presumably you didnt model the second conformation unless there was some
>> feature in an earlier map to suggest it existed?
>> Good luck Eleanor
>>
>>
>> On Wed, 6 Sept 2023 at 03:18, Pavel Afonine  wrote:
>>
>>> Hi Matt,
>>> I believe figure 3 here:
>>> https://www.nature.com/articles/s41467-018-06957-w
>>> is relevant to your question.
>>> Pavel
>>>
>>>
>>> On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod 
>>> wrote:
>>>
 Hi all,

 I am trying to get some insight in the accuracy/precision of occupancy
 refinements.  I have done some 2-state occupancy refinements and have
 observed the refinement achieving ~0.25-0.3 occupancy for the minor
 population.  This population, when observing the electron density maps, had
 essentially no evidence for it being present.  I was wondering:

 What are the errors in the reported occupancies?

 Is there a lower and upper limit to occupancy refinements?  As in, if
 you occupancy refine two states and one is imaginary will it refine to
 approximately 1 and 0?  Or does the background noise always given a
 positive number to the imaginary set?  This would, to me at least, be the
 lower and upper limits to the occupancy refinements and could be used as a
 normalization factor for other atoms.  Maybe my logic is off...

 Any insight or literature would be appreciated!
 Matt

 

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Re: [ccp4bb] Occupancy Refinement limitation

[a . perrakis]

I would be tempted to try phenix.ensemble_refinement instead.

Look at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949522/
or 
https://phenix-online.org/phenixwebsite_static/mainsite/files/presentations/ensemble_refinement_burnley_at_al_10DEC2012.pdf
or https://elifesciences.org/articles/00311

Tassos

On 6 Sep 2023, at 12:37, Matt Mcleod 
mailto:mjmcleo...@gmail.com>> wrote:

Hi,

I should be a bit more specific.

We have many crystal structures to indicate that a loop adopts conformation A 
and conformation B, or - the loop can be disordered where the electron density 
is washed out.  These states are dependent on how we perturb the system.

We have a series of data, as a function of osmolyte concentration, that adjusts 
this equilibrium and we want to put a numerical value onto it.  Resolution is 
between 1.7 - 2.0 A. This is non-anomalous data but certainly would be helpful 
in the future to include if possible some anomalous scatters. Have been using 
phenix.refine.

So what we have done is modeled both conformation A and B and are refining the 
data in order to get the occupancies (fixing B-factors) regardless of if there 
is electron density present for one of the two conformations since in some 
cases (likely all) there will be a population of both A, B, and disordered and 
the relative true occupancies will move.  I am trying to sort out how accurate 
these occupancy values are as opposed to showing the electron density for each 
conformation fit at some common threshold.  My general sense is that if it is 
modeled but no electron density, there will be a non-zero value and vice versa 
if it is the only conformation present it will be less than unity.

I will take a look at the references!  Very much appreciated.
Matt

On Wed, 6 Sept 2023 at 02:08, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Well - occupancy refinement is particularly imprecise, and highly correlated 
with temperature factors.
Also the population for a surface ARG or LYS may well have more than two 
conformations, whereas some internal residue is better defined.
There is also the Q of solvent - dual occupancies will generate dual solvent 
networks..

..You dont say what resolution your data are. Again at 0.8A you can be 
confident - at 3A it is at best a guess. So I for one do not take the numbers 
very seriously - they are a flag only.

Presumably you didnt model the second conformation unless there was some 
feature in an earlier map to suggest it existed?
Good luck Eleanor


On Wed, 6 Sept 2023 at 03:18, Pavel Afonine 
mailto:pafon...@gmail.com>> wrote:
Hi Matt,
I believe figure 3 here:
https://www.nature.com/articles/s41467-018-06957-w
is relevant to your question.
Pavel


On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod 
mailto:mjmcleo...@gmail.com>> wrote:
Hi all,

I am trying to get some insight in the accuracy/precision of occupancy 
refinements.  I have done some 2-state occupancy refinements and have observed 
the refinement achieving ~0.25-0.3 occupancy for the minor population.  This 
population, when observing the electron density maps, had essentially no 
evidence for it being present.  I was wondering:

What are the errors in the reported occupancies?

Is there a lower and upper limit to occupancy refinements?  As in, if you 
occupancy refine two states and one is imaginary will it refine to 
approximately 1 and 0?  Or does the background noise always given a positive 
number to the imaginary set?  This would, to me at least, be the lower and 
upper limits to the occupancy refinements and could be used as a normalization 
factor for other atoms.  Maybe my logic is off...

Any insight or literature would be appreciated!
Matt



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--
Matthew Jordan McLeod, PhD
Post-Doctoral Fellow - Cornell University






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This message 

Re: [ccp4bb] Occupancy Refinement limitation

[Matt Mcleod]

Hi,

I should be a bit more specific.

We have many crystal structures to indicate that a loop adopts conformation
A and conformation B, or - the loop can be disordered where the electron
density is washed out.  These states are dependent on how we perturb the
system.

We have a series of data, as a function of osmolyte concentration, that
adjusts this equilibrium and we want to put a numerical value onto it.
Resolution is between 1.7 - 2.0 A. This is non-anomalous data but certainly
would be helpful in the future to include if possible some anomalous
scatters. Have been using phenix.refine.

So what we have done is modeled both conformation A and B and are refining
the data in order to get the occupancies (fixing B-factors) regardless of
if there is electron density present for one of the two conformations since
in some cases (likely all) there will be a population of both A, B, and
disordered and the relative true occupancies will move.  I am trying to
sort out how accurate these occupancy values are as opposed to showing the
electron density for each conformation fit at some common threshold.  My
general sense is that if it is modeled but no electron density, there will
be a non-zero value and vice versa if it is the only conformation present
it will be less than unity.

I will take a look at the references!  Very much appreciated.
Matt

On Wed, 6 Sept 2023 at 02:08, Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> Well - occupancy refinement is particularly imprecise, and highly
> correlated with temperature factors.
> Also the population for a surface ARG or LYS may well have more than two
> conformations, whereas some internal residue is better defined.
> There is also the Q of solvent - dual occupancies will generate dual
> solvent networks..
>
> ..You dont say what resolution your data are. Again at 0.8A you can be
> confident - at 3A it is at best a guess. So I for one do not take the
> numbers very seriously - they are a flag only.
>
> Presumably you didnt model the second conformation unless there was some
> feature in an earlier map to suggest it existed?
> Good luck Eleanor
>
>
> On Wed, 6 Sept 2023 at 03:18, Pavel Afonine  wrote:
>
>> Hi Matt,
>> I believe figure 3 here:
>> https://www.nature.com/articles/s41467-018-06957-w
>> is relevant to your question.
>> Pavel
>>
>>
>> On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod  wrote:
>>
>>> Hi all,
>>>
>>> I am trying to get some insight in the accuracy/precision of occupancy
>>> refinements.  I have done some 2-state occupancy refinements and have
>>> observed the refinement achieving ~0.25-0.3 occupancy for the minor
>>> population.  This population, when observing the electron density maps, had
>>> essentially no evidence for it being present.  I was wondering:
>>>
>>> What are the errors in the reported occupancies?
>>>
>>> Is there a lower and upper limit to occupancy refinements?  As in, if
>>> you occupancy refine two states and one is imaginary will it refine to
>>> approximately 1 and 0?  Or does the background noise always given a
>>> positive number to the imaginary set?  This would, to me at least, be the
>>> lower and upper limits to the occupancy refinements and could be used as a
>>> normalization factor for other atoms.  Maybe my logic is off...
>>>
>>> Any insight or literature would be appreciated!
>>> Matt
>>>
>>> 
>>>
>>> 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/
>>>
>>
>> --
>>
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>
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-- 
*Matthew Jordan McLeod, PhD*
*Post-Doctoral Fellow - Cornell University*



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Re: [ccp4bb] Occupancy Refinement limitation

[John R Helliwell]

Dear Matt,I believe this study we made may well assist your evaluations:-(IUCr) Experience with exchange and archiving of raw data: comparison of data from two diffractometers and four software packages on a series of lysozyme crystalsjournals.iucr.orgBest wishes,John Emeritus Professor John R Helliwell DScOn 5 Sep 2023, at 19:32, Matt McLeod  wrote:Hi all,I am trying to get some insight in the accuracy/precision of occupancy refinements.  I have done some 2-state occupancy refinements and have observed the refinement achieving ~0.25-0.3 occupancy for the minor population.  This population, when observing the electron density maps, had essentially no evidence for it being present.  I was wondering:What are the errors in the reported occupancies?Is there a lower and upper limit to occupancy refinements?  As in, if you occupancy refine two states and one is imaginary will it refine to approximately 1 and 0?  Or does the background noise always given a positive number to the imaginary set?  This would, to me at least, be the lower and upper limits to the occupancy refinements and could be used as a normalization factor for other atoms.  Maybe my logic is off...Any insight or literature would be appreciated!MattTo unsubscribe from the CCP4BB list, click the following link:https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1This 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/

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Re: [ccp4bb] Occupancy Refinement limitation

[Tim Gruene]

Hi Matt,

this can be done with SHELXL, even at 2-3A resolution. Anomalous data
do help reduce the correlation between B-factor and occupancy 
(see e.g. https://doi.org/10.1126/science.1254840 to stick with Pavel's
example)

SHELXL prints the correlation coefficient between occupancy and
b-factor, and also the ESD of the occupancy. The is more reliable with
least-square refinement, but for large structures, you may need a lot
of RAM.

PDB2INS (https://www.nature.com/articles/s41467-018-06957-w)
conveniently prepares the INS-file from your PDB for you.

Best wishes,
Tim

On Tue, 5 Sep 2023 19:31:25 +0100 Matt McLeod 
wrote:

> Hi all,
> 
> I am trying to get some insight in the accuracy/precision of
> occupancy refinements.  I have done some 2-state occupancy
> refinements and have observed the refinement achieving ~0.25-0.3
> occupancy for the minor population.  This population, when observing
> the electron density maps, had essentially no evidence for it being
> present.  I was wondering:
> 
> What are the errors in the reported occupancies?
> 
> Is there a lower and upper limit to occupancy refinements?  As in, if
> you occupancy refine two states and one is imaginary will it refine
> to approximately 1 and 0?  Or does the background noise always given
> a positive number to the imaginary set?  This would, to me at least,
> be the lower and upper limits to the occupancy refinements and could
> be used as a normalization factor for other atoms.  Maybe my logic is
> off...
> 
> Any insight or literature would be appreciated!
> Matt
> 
> 
> 
> 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
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-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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pgp3Nciy9jGRf.pgp
Description: OpenPGP digital signature

Re: [ccp4bb] Occupancy Refinement limitation

[Eleanor Dodson]

Well - occupancy refinement is particularly imprecise, and highly
correlated with temperature factors.
Also the population for a surface ARG or LYS may well have more than two
conformations, whereas some internal residue is better defined.
There is also the Q of solvent - dual occupancies will generate dual
solvent networks..

..You dont say what resolution your data are. Again at 0.8A you can be
confident - at 3A it is at best a guess. So I for one do not take the
numbers very seriously - they are a flag only.

Presumably you didnt model the second conformation unless there was some
feature in an earlier map to suggest it existed?
Good luck Eleanor


On Wed, 6 Sept 2023 at 03:18, Pavel Afonine  wrote:

> Hi Matt,
> I believe figure 3 here:
> https://www.nature.com/articles/s41467-018-06957-w
> is relevant to your question.
> Pavel
>
>
> On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod  wrote:
>
>> Hi all,
>>
>> I am trying to get some insight in the accuracy/precision of occupancy
>> refinements.  I have done some 2-state occupancy refinements and have
>> observed the refinement achieving ~0.25-0.3 occupancy for the minor
>> population.  This population, when observing the electron density maps, had
>> essentially no evidence for it being present.  I was wondering:
>>
>> What are the errors in the reported occupancies?
>>
>> Is there a lower and upper limit to occupancy refinements?  As in, if you
>> occupancy refine two states and one is imaginary will it refine to
>> approximately 1 and 0?  Or does the background noise always given a
>> positive number to the imaginary set?  This would, to me at least, be the
>> lower and upper limits to the occupancy refinements and could be used as a
>> normalization factor for other atoms.  Maybe my logic is off...
>>
>> Any insight or literature would be appreciated!
>> Matt
>>
>> 
>>
>> 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/
>>
>
> --
>
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Re: [ccp4bb] Occupancy Refinement limitation

[Pavel Afonine]

Hi Matt,
I believe figure 3 here:
https://www.nature.com/articles/s41467-018-06957-w
is relevant to your question.
Pavel


On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod  wrote:

> Hi all,
>
> I am trying to get some insight in the accuracy/precision of occupancy
> refinements.  I have done some 2-state occupancy refinements and have
> observed the refinement achieving ~0.25-0.3 occupancy for the minor
> population.  This population, when observing the electron density maps, had
> essentially no evidence for it being present.  I was wondering:
>
> What are the errors in the reported occupancies?
>
> Is there a lower and upper limit to occupancy refinements?  As in, if you
> occupancy refine two states and one is imaginary will it refine to
> approximately 1 and 0?  Or does the background noise always given a
> positive number to the imaginary set?  This would, to me at least, be the
> lower and upper limits to the occupancy refinements and could be used as a
> normalization factor for other atoms.  Maybe my logic is off...
>
> Any insight or literature would be appreciated!
> Matt
>
> 
>
> 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/
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[ccp4bb] Occupancy Refinement limitation

[Matt McLeod]

Hi all,

I am trying to get some insight in the accuracy/precision of occupancy 
refinements.  I have done some 2-state occupancy refinements and have observed 
the refinement achieving ~0.25-0.3 occupancy for the minor population.  This 
population, when observing the electron density maps, had essentially no 
evidence for it being present.  I was wondering:

What are the errors in the reported occupancies?

Is there a lower and upper limit to occupancy refinements?  As in, if you 
occupancy refine two states and one is imaginary will it refine to 
approximately 1 and 0?  Or does the background noise always given a positive 
number to the imaginary set?  This would, to me at least, be the lower and 
upper limits to the occupancy refinements and could be used as a normalization 
factor for other atoms.  Maybe my logic is off...

Any insight or literature would be appreciated!
Matt



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Herman . Schreuder]

Dear HK,

I agree with Artem, I would put the ketone in the green density below in your 
pictures and make a covalent link with the arginine. The single oxygen I would 
put in the density currently occupied by the ketone and add one water there as 
well. In your refined omit maps, the current ketone density is rather weak and 
weakly connected with the ligand. This is more reminiscent of a bound water 
than of a covalent part of the ligand.

Best,
Herman

-Ursprüngliche Nachricht-
Von: t...@em.uni-frankfurt.de  
Gesendet: Donnerstag, 7. November 2019 13:51
An: Dale Tronrud ; Seijo, Jose A. 
; Schreuder, Herman /DE 

Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron 
density of a residue and ligand

EXTERNAL : Real sender is  prvs=1214477c0d=t...@em.uni-frankfurt.de   



Dear Dale, Jose, Herman,

For Dale,
The contour level of residual positive density in the map is 0.28
e/A^3 and 3 sigma as shown in Coot, when it is refined in full occupancy. I 
tried to model the amino-sugar (flip the ligand) to the mysterious density but 
it has no space for it as it clashes with the surrounding amino acids.

For Herman,
I noticed that there are some impurities of the ligand from the literature and 
it also mentions that the ligand tends to be degraded due to pH and oxidation. 
It might be a degraded product.

For Jose,
I tried to refine it with one water molecule instead of two (Figure - 
fullocc1.png and fullocc2.png. They are the same but with different
view) and positive density seems to be reduced with full occupancy refinement 
of ligand and water. I also performed a refinement with occupancy refinement 
with ligand and water
(occupancy_ligand_water.png) but it seems to me that positive density appears 
again in comparison with full occupancy refinement of water.  
But the B-factor of water is too low if refine it with a occupancy refinement. 
If occupancy of water is set to 1 then the B-factor seems to be correlated well 
with the surroundings.

Thanks

Best
HK


Quoting Dale Tronrud :

> Hi,
>
>I'm curious.  What is the contour level of the residual positive 
> density in the maps with full occupancy ligand and Arg, and how does 
> that level compare to the level of the contour of your omit map?  
> e/A^3 would be the most useful.
>
>Without the contour levels it isn't possible to distinguish between 
> the absence of fully occupied atoms and something more minor.
>
>Could your ligand be spending a small part of its time flipped over 
> on the long axis of your fused ring system?  This would put the 
> sugar-like group over by your mystery density.
>
> Dale Tronrud
>
> On 11/6/2019 10:58 PM, Heng-Keat Tam wrote:
>> Hi Herman,
>>
>> Thanks for the suggestion. In principle, I have refined the structure 
>> without Arg and ligand, as well as the connected residues to the Arg.
>> However, I still see the connected density of Arg and the ligand 
>> (Figure
>> - unmodel1.png - only removal of Arg and ligand).
>>
>> I have tried to refine the degraded product but the positive density 
>> in between Arg and the ligand is still there, indicating something 
>> should be modeled there (Figure - degraded_product.png and 
>> degraded_product2.png - both figures are the same ligand but with 
>> different side view).
>>
>> I also tried to model the ligand in different conformation but still 
>> the same as shown in degraded product. The positive density is there 
>> between Arg and the ligand.
>>
>> For both cases, I refined Arg with full occupancy but ligand with 
>> occupancy refinement. Now, Arg did not flip away from the density.
>>
>> I might have a look at the mass-spec to check whether it is 
>> covalently linked although I am not aware of the reactivity of the 
>> moiety of the ligand and we never see this compound to covalent link 
>> with protein in our experience.
>>
>> Thanks for the advice and suggestion. It is very helpful.
>>
>> Thanks.
>>
>> Best
>> HK
>>
>>
>>
>> Quoting herman.schreu...@sanofi.com:
>>
>>> Hi Heng-Keat,
>>>
>>> I had again a look at your electron density pictures and these were 
>>> my
>>> impressions:
>>> 1) the Arginine as fitted looks fully occupied. There are no hints 
>>> for an alternative conformations.
>>> 2) The fit of the ligand, although reasonable, is not extremely good.
>>>
>>> It seems that the only reason to invoke partial occupancies for the 
>>> Arginine and ligand are problems to fit both the ligand and the 
>>> arginine in the available density. This means that for the 
>>> overlapping parts, to sum of the occupancies 

Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Artem Evdokimov]

I am willing to bet that the ketone is covalently reacting with the
arginine and the result is what you see :)

Artem

- Cosmic Cats approve of this message


On Thu, Nov 7, 2019 at 3:33 AM  wrote:

> Dear HK,
>
> In your case mass spec would be extremely valuable. Not only could it
> prove or rule out a covalent link, it will also convince referees that the
> link is real.
> There is clearly more density than the degraded product you showed. I
> would also ask an experienced chemist if a link between your ligand and the
> arginine would be possible. Finally, I would also do a quality check on the
> ligand you used. Might it be that some reactive intermediate from the
> synthesis still is present?
>
> Best,
> Herman
>
> -Ursprüngliche Nachricht-
> Von: t...@em.uni-frankfurt.de 
> Gesendet: Donnerstag, 7. November 2019 07:58
> An: Schreuder, Herman /DE 
> Cc: CCP4BB@jiscmail.ac.uk
> Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping
> electron density of a residue and ligand
>
> EXTERNAL : Real sender is  prvs=1214477c0d=t...@em.uni-frankfurt.de
>
>
>
> Hi Herman,
>
> Thanks for the suggestion. In principle, I have refined the structure
> without Arg and ligand, as well as the connected residues to the Arg.
> However, I still see the connected density of Arg and the ligand (Figure -
> unmodel1.png - only removal of Arg and ligand).
>
> I have tried to refine the degraded product but the positive density in
> between Arg and the ligand is still there, indicating something should be
> modeled there (Figure - degraded_product.png and degraded_product2.png -
> both figures are the same ligand but with different side view).
>
> I also tried to model the ligand in different conformation but still the
> same as shown in degraded product. The positive density is there between
> Arg and the ligand.
>
> For both cases, I refined Arg with full occupancy but ligand with
> occupancy refinement. Now, Arg did not flip away from the density.
>
> I might have a look at the mass-spec to check whether it is covalently
> linked although I am not aware of the reactivity of the moiety of the
> ligand and we never see this compound to covalent link with protein in our
> experience.
>
> Thanks for the advice and suggestion. It is very helpful.
>
> Thanks.
>
> Best
> HK
>
>
>
> Quoting herman.schreu...@sanofi.com:
>
> > Hi Heng-Keat,
> >
> > I had again a look at your electron density pictures and these were my
> > impressions:
> > 1) the Arginine as fitted looks fully occupied. There are no hints for
> > an alternative conformations.
> > 2) The fit of the ligand, although reasonable, is not extremely good.
> >
> > It seems that the only reason to invoke partial occupancies for the
> > Arginine and ligand are problems to fit both the ligand and the
> > arginine in the available density. This means that for the overlapping
> > parts, to sum of the occupancies is maximal 1.0.
> > However, the ligand and the Arginine do not look half occupied.
> >
> > So I would look at other explanations:
> > - would it be possible that the ligand reacts covalently with the
> > Arginine? The density looks pretty continuous to me.
> > - could it be that instead of the intended ligand, some side- or
> > degradation product has bound?
> >
> > What I would also do, but what you probably already did, is to delete
> > the ligand and the Arginine side chain atoms and run several rounds of
> > refinement, to get an electron density map with as much bias as
> > possible removed.
> >
> > Good luck!
> > Herman
> >
> > -Original Message-
> > From: CCP4 bulletin board  On Behalf Of
> > Heng-Keat Tam
> > Sent: Mittwoch, 6. November 2019 07:30
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping
> > electron density of a residue and ligand
> >
> > EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk
> >
> >
> >
> > Hi Nestor,
> >
> > I think the reason for Arg side chain to curl up is because I refined
> > ligand and Arg side chain with occupancy refinement, and the Arg moved
> > away from the density, most likely due to 'repulsion' from ligand.
> >
> > The other question is: Is it possible that the H-bonds stay very
> > close? As I tried to 'real space refine' in coot, and the Arg side
> > chain flipped away from the density.
> >
> > Thanks for the suggestion.
> >
> > Best
> > HK
> >
> >
> > Quoting Nestor Concha :
> >
> >> Hi Tam,
> >> T

[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Herman . Schreuder]

Dear HK,

In your case mass spec would be extremely valuable. Not only could it prove or 
rule out a covalent link, it will also convince referees that the link is real.
There is clearly more density than the degraded product you showed. I would 
also ask an experienced chemist if a link between your ligand and the arginine 
would be possible. Finally, I would also do a quality check on the ligand you 
used. Might it be that some reactive intermediate from the synthesis still is 
present?

Best,
Herman

-Ursprüngliche Nachricht-
Von: t...@em.uni-frankfurt.de  
Gesendet: Donnerstag, 7. November 2019 07:58
An: Schreuder, Herman /DE 
Cc: CCP4BB@jiscmail.ac.uk
Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron 
density of a residue and ligand

EXTERNAL : Real sender is  prvs=1214477c0d=t...@em.uni-frankfurt.de   



Hi Herman,

Thanks for the suggestion. In principle, I have refined the structure without 
Arg and ligand, as well as the connected residues to the Arg.  
However, I still see the connected density of Arg and the ligand (Figure - 
unmodel1.png - only removal of Arg and ligand).

I have tried to refine the degraded product but the positive density in between 
Arg and the ligand is still there, indicating something should be modeled there 
(Figure - degraded_product.png and degraded_product2.png - both figures are the 
same ligand but with different side view).

I also tried to model the ligand in different conformation but still the same 
as shown in degraded product. The positive density is there between Arg and the 
ligand.

For both cases, I refined Arg with full occupancy but ligand with occupancy 
refinement. Now, Arg did not flip away from the density.

I might have a look at the mass-spec to check whether it is covalently linked 
although I am not aware of the reactivity of the moiety of the ligand and we 
never see this compound to covalent link with protein in our experience.

Thanks for the advice and suggestion. It is very helpful.

Thanks.

Best
HK



Quoting herman.schreu...@sanofi.com:

> Hi Heng-Keat,
>
> I had again a look at your electron density pictures and these were my 
> impressions:
> 1) the Arginine as fitted looks fully occupied. There are no hints for 
> an alternative conformations.
> 2) The fit of the ligand, although reasonable, is not extremely good.
>
> It seems that the only reason to invoke partial occupancies for the 
> Arginine and ligand are problems to fit both the ligand and the 
> arginine in the available density. This means that for the overlapping 
> parts, to sum of the occupancies is maximal 1.0.
> However, the ligand and the Arginine do not look half occupied.
>
> So I would look at other explanations:
> - would it be possible that the ligand reacts covalently with the 
> Arginine? The density looks pretty continuous to me.
> - could it be that instead of the intended ligand, some side- or 
> degradation product has bound?
>
> What I would also do, but what you probably already did, is to delete 
> the ligand and the Arginine side chain atoms and run several rounds of 
> refinement, to get an electron density map with as much bias as 
> possible removed.
>
> Good luck!
> Herman
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of 
> Heng-Keat Tam
> Sent: Mittwoch, 6. November 2019 07:30
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping 
> electron density of a residue and ligand
>
> EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk
>
>
>
> Hi Nestor,
>
> I think the reason for Arg side chain to curl up is because I refined 
> ligand and Arg side chain with occupancy refinement, and the Arg moved 
> away from the density, most likely due to 'repulsion' from ligand.
>
> The other question is: Is it possible that the H-bonds stay very 
> close? As I tried to 'real space refine' in coot, and the Arg side 
> chain flipped away from the density.
>
> Thanks for the suggestion.
>
> Best
> HK
>
>
> Quoting Nestor Concha :
>
>> Hi Tam,
>> The density looks very strong and therefore I'm going to guess that 
>> the Arg guanidimium stays in contact/interacts with the ligand and 
>> with the phosphate/sulfate next to it. Perhaps it is one of those 
>> close interactions with shorter H-bonds that usual given the 
>> arrangement of ligand-phosphate/sulfate-Arg.  I'd try to find a 
>> rotamer for the Arg that leaves the interactions intact rather than 
>> refine occupancies. Seems that the Arg side chain is curled up 
>> Nestor
>>
>> -Original Message-
>> From: CCP4 bulletin board  On Behalf Of 
>> Heng-Keat Tam
>> Sent: Tuesday, November 5, 2019 10:55 AM
>> To: CCP4BB@JISCMAIL.AC.UK
>

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Herman . Schreuder]

Hi Heng-Keat,

I had again a look at your electron density pictures and these were my 
impressions:
1) the Arginine as fitted looks fully occupied. There are no hints for an 
alternative conformations. 
2) The fit of the ligand, although reasonable, is not extremely good.

It seems that the only reason to invoke partial occupancies for the Arginine 
and ligand are problems to fit both the ligand and the arginine in the 
available density. This means that for the overlapping parts, to sum of the 
occupancies is maximal 1.0. However, the ligand and the Arginine do not look 
half occupied.

So I would look at other explanations: 
- would it be possible that the ligand reacts covalently with the Arginine? The 
density looks pretty continuous to me.
- could it be that instead of the intended ligand, some side- or degradation 
product has bound?

What I would also do, but what you probably already did, is to delete the 
ligand and the Arginine side chain atoms and run several rounds of refinement, 
to get an electron density map with as much bias as possible removed.

Good luck!
Herman

-Original Message-
From: CCP4 bulletin board  On Behalf Of Heng-Keat Tam
Sent: Mittwoch, 6. November 2019 07:30
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron 
density of a residue and ligand

EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk   



Hi Nestor,

I think the reason for Arg side chain to curl up is because I refined ligand 
and Arg side chain with occupancy refinement, and the Arg moved away from the 
density, most likely due to 'repulsion' from ligand.

The other question is: Is it possible that the H-bonds stay very close? As I 
tried to 'real space refine' in coot, and the Arg side chain flipped away from 
the density.

Thanks for the suggestion.

Best
HK


Quoting Nestor Concha :

> Hi Tam,
> The density looks very strong and therefore I'm going to guess that 
> the Arg guanidimium stays in contact/interacts with the ligand and 
> with the phosphate/sulfate next to it. Perhaps it is one of those 
> close interactions with shorter H-bonds that usual given the 
> arrangement of ligand-phosphate/sulfate-Arg.  I'd try to find a 
> rotamer for the Arg that leaves the interactions intact rather than 
> refine occupancies. Seems that the Arg side chain is curled up 
> Nestor
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of 
> Heng-Keat Tam
> Sent: Tuesday, November 5, 2019 10:55 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Occupancy refinement of overlapping electron 
> density of a residue and ligand
>
> EXTERNAL
>
> Dear Rob,
>
> I would like to model the alternative position for the side chain of
> R120 but I don't really know whether the alternative conformation 
> exist as shown in the attached figure - density without the ligand and
> R120 overlaid with the refined structures of modeled ligand and R120.
> The ligand was modeled in two different conformations. From the 
> density, it seems to me that the density is connected or overlapped.
>
> It should not be a post-translation modification as it is well-known 
> that there is no post-translation modification for this protein.
> Furthermore, the crystal was obtained by co-crystallization of protein 
> with the ligand itself. The density seems to be the expected ligand.
>
> Thanks for the advice.
>
> Best regards
> HK
>
>
> Quoting Robert Nicholls :
>
>> Dear HK,
>>
>> No that's not quite correct - 'occupancy group alts complete' means 
>> that both R120 and the ligand are constrained so that their 
>> occupancies sum to unity. In contrast, 'occupancy group alts 
>> incomplete' means that the occupancies of R120 and the ligand will 
>> not be constrained to sum to unity (but the sum of their occupancies 
>> must be less than one). In both cases, R120 and the ligand will "see" 
>> each other in a certain sense. But, because they are assigned to 
>> different groups, it is assumed that they are present in different 
>> parts of the crystal. This means that they can overlap.
>>
>> Assuming that the ligand is in the correct conformation, I suspect 
>> the source of your problem is that you are modelling the side chain 
>> of
>> R120 as only one conformation. And I would also include the other 
>> atoms in the side chain - CB and CG.
>>
>> If you are modelling the sidechain of R120 with partial occupancies, 
>> then you should model those side chain atoms in two alternative 
>> positions (i.e. representing the portions of the crystal that 
>> do/don't have the ligand bound). This will help to ensure that your 
>> model makes physical sense. So the ligand plus the alt of R120 in the 

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Heng-Keat Tam]

Hi Nestor,

I think the reason for Arg side chain to curl up is because I refined  
ligand and Arg side chain with occupancy refinement, and the Arg moved  
away from the density, most likely due to 'repulsion' from ligand.


The other question is: Is it possible that the H-bonds stay very  
close? As I tried to 'real space refine' in coot, and the Arg side  
chain flipped away from the density.


Thanks for the suggestion.

Best
HK


Quoting Nestor Concha :


Hi Tam,
The density looks very strong and therefore I'm going to guess that  
the Arg guanidimium stays in contact/interacts with the ligand and  
with the phosphate/sulfate next to it. Perhaps it is one of those  
close interactions with shorter H-bonds that usual given the  
arrangement of ligand-phosphate/sulfate-Arg.  I'd try to find a  
rotamer for the Arg that leaves the interactions intact rather than  
refine occupancies. Seems that the Arg side chain is curled up 

Nestor

-Original Message-
From: CCP4 bulletin board  On Behalf Of Heng-Keat Tam
Sent: Tuesday, November 5, 2019 10:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Occupancy refinement of overlapping electron  
density of a residue and ligand


EXTERNAL

Dear Rob,

I would like to model the alternative position for the side chain of
R120 but I don't really know whether the alternative conformation  
exist as shown in the attached figure - density without the ligand and

R120 overlaid with the refined structures of modeled ligand and R120.
The ligand was modeled in two different conformations. From the  
density, it seems to me that the density is connected or overlapped.


It should not be a post-translation modification as it is well-known  
that there is no post-translation modification for this protein.
Furthermore, the crystal was obtained by co-crystallization of  
protein with the ligand itself. The density seems to be the expected  
ligand.


Thanks for the advice.

Best regards
HK


Quoting Robert Nicholls :


Dear HK,

No that's not quite correct - 'occupancy group alts complete' means
that both R120 and the ligand are constrained so that their
occupancies sum to unity. In contrast, 'occupancy group alts
incomplete' means that the occupancies of R120 and the ligand will not
be constrained to sum to unity (but the sum of their occupancies must
be less than one). In both cases, R120 and the ligand will "see" each
other in a certain sense. But, because they are assigned to different
groups, it is assumed that they are present in different parts of the
crystal. This means that they can overlap.

Assuming that the ligand is in the correct conformation, I suspect the
source of your problem is that you are modelling the side chain of
R120 as only one conformation. And I would also include the other
atoms in the side chain - CB and CG.

If you are modelling the sidechain of R120 with partial occupancies,
then you should model those side chain atoms in two alternative
positions (i.e. representing the portions of the crystal that do/don't
have the ligand bound). This will help to ensure that your model makes
physical sense. So the ligand plus the alt of R120 in the portion of
the crystal that contains the ligand would be assigned to one
occupancy group, and the alt of R120 in the portion of the crystal
that does not contain the ligand would be assigned to the second
group. In this case it would be appropriate to specify 'occupancy
group alts complete', because the occupancies for the two alternative
conformers of R120 should sum to unity. Although no doubt the
estimation of the occupancies would be dominated by the ligand in this
case.

Be sure to check your B-factors after occupancy refinement to make
sure the whole picture makes sense. Assuming your current model is
essentially correct, from visual inspection it looks like R120 will
have low occupancy and low B-factors when the ligand is not present
(or at least B-factors consistent with the environment), but will have
high occupancy and high B-factors when the ligand is present.

On another note, have you considered whether that part of the ligand
could be modelled in a slightly different conformation, or whether
there could be a post-translation modification?

I hope that helps,
Rob


Dr Rob Nicholls
Senior Investigator Scientist
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH




On 5 Nov 2019, at 13:36, HK  wrote:

Dear all,

I have problem with occupancy refinement by Refmac5 for an
overlapping electron density of part of residue (an arginine) and
part of ligand (tetracyclic compound) (attached figures in Dropbox
with a link as shown below).

https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?
dl=0

I refined part of the side chain of residue 120 and ligand (chain J
residue 1105) with Refmac keyword as shown below. Unfortunately, the
side chain of arginine moves away from the density but the ligand
stays in the density. A

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Robert Nicholls]

Dear HK,

If you believe that R120 only adopts one conformation then you should not 
refine the occupancies of the side chain atoms. It makes no physical sense to 
have occupancies less than one for these atoms, but occupancy equal to one for 
the rest of the protein (unless you believe the side chain to be cleaved, or 
otherwise chemically modified). However, occupancy refinement of the ligand 
itself can be justified by partial binding.

Best regards,
Rob


> On 5 Nov 2019, at 15:55, t...@em.uni-frankfurt.de wrote:
> 
> Dear Rob,
> 
> I would like to model the alternative position for the side chain of R120 but 
> I don't really know whether the alternative conformation exist as shown in 
> the attached figure - density without the ligand and R120 overlaid with the 
> refined structures of modeled ligand and R120. The ligand was modeled in two 
> different conformations. From the density, it seems to me that the density is 
> connected or overlapped.
> 
> It should not be a post-translation modification as it is well-known that 
> there is no post-translation modification for this protein. Furthermore, the 
> crystal was obtained by co-crystallization of protein with the ligand itself. 
> The density seems to be the expected ligand.
> 
> Thanks for the advice.
> 
> Best regards
> HK
> 
> 
> Quoting Robert Nicholls :
> 
>> Dear HK,
>> 
>> No that's not quite correct - 'occupancy group alts complete' means that 
>> both R120 and the ligand are constrained so that their occupancies sum to 
>> unity. In contrast, 'occupancy group alts incomplete' means that the 
>> occupancies of R120 and the ligand will not be constrained to sum to unity 
>> (but the sum of their occupancies must be less than one). In both cases, 
>> R120 and the ligand will "see" each other in a certain sense. But, because 
>> they are assigned to different groups, it is assumed that they are present 
>> in different parts of the crystal. This means that they can overlap.
>> 
>> Assuming that the ligand is in the correct conformation, I suspect the 
>> source of your problem is that you are modelling the side chain of R120 as 
>> only one conformation. And I would also include the other atoms in the side 
>> chain - CB and CG.
>> 
>> If you are modelling the sidechain of R120 with partial occupancies, then 
>> you should model those side chain atoms in two alternative positions (i.e. 
>> representing the portions of the crystal that do/don't have the ligand 
>> bound). This will help to ensure that your model makes physical sense. So 
>> the ligand plus the alt of R120 in the portion of the crystal that contains 
>> the ligand would be assigned to one occupancy group, and the alt of R120 in 
>> the portion of the crystal that does not contain the ligand would be 
>> assigned to the second group. In this case it would be appropriate to 
>> specify 'occupancy group alts complete', because the occupancies for the two 
>> alternative conformers of R120 should sum to unity. Although no doubt the 
>> estimation of the occupancies would be dominated by the ligand in this case.
>> 
>> Be sure to check your B-factors after occupancy refinement to make sure the 
>> whole picture makes sense. Assuming your current model is essentially 
>> correct, from visual inspection it looks like R120 will have low occupancy 
>> and low B-factors when the ligand is not present (or at least B-factors 
>> consistent with the environment), but will have high occupancy and high 
>> B-factors when the ligand is present.
>> 
>> On another note, have you considered whether that part of the ligand could 
>> be modelled in a slightly different conformation, or whether there could be 
>> a post-translation modification?
>> 
>> I hope that helps,
>> Rob
>> 
>> 
>> Dr Rob Nicholls
>> Senior Investigator Scientist
>> MRC Laboratory of Molecular Biology
>> Francis Crick Avenue
>> Cambridge Biomedical Campus
>> Cambridge CB2 0QH
>> 
>> 
>> 
>>> On 5 Nov 2019, at 13:36, HK  wrote:
>>> 
>>> Dear all,
>>> 
>>> I have problem with occupancy refinement by Refmac5 for an overlapping 
>>> electron density of part of residue (an arginine) and part of ligand 
>>> (tetracyclic compound) (attached figures in Dropbox with a link as shown 
>>> below).
>>> 
>>> https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0
>>> 
>>> I refined part of the side chain of residue 120 and ligand (chain J residue 
>>> 1105) with Refmac keyword as shown below. Unfortunately, the side chain of 
>>> arginine moves away from the density but the ligand stays in the density. 
>>> As far as I understood, occupancy refinement with keyword 'occupancy group 
>>> alts complete' means both R120 and the ligand do not meet each other.  Did 
>>> I miss something from the occupancy refinement keyword?
>>> 
>>> occupancy group id 1 chain A residue 120 atom NE
>>> occupancy group id 1 chain A residue 120 atom CZ
>>> occupancy group id 1 chain A residue 120 atom NH2
>>> occupancy group id 1 chain A 

Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Robert Nicholls]

Dear HK,

No that's not quite correct - 'occupancy group alts complete' means that both 
R120 and the ligand are constrained so that their occupancies sum to unity. In 
contrast, 'occupancy group alts incomplete' means that the occupancies of R120 
and the ligand will not be constrained to sum to unity (but the sum of their 
occupancies must be less than one). In both cases, R120 and the ligand will 
"see" each other in a certain sense. But, because they are assigned to 
different groups, it is assumed that they are present in different parts of the 
crystal. This means that they can overlap.

Assuming that the ligand is in the correct conformation, I suspect the source 
of your problem is that you are modelling the side chain of R120 as only one 
conformation. And I would also include the other atoms in the side chain - CB 
and CG.

If you are modelling the sidechain of R120 with partial occupancies, then you 
should model those side chain atoms in two alternative positions (i.e. 
representing the portions of the crystal that do/don't have the ligand bound). 
This will help to ensure that your model makes physical sense. So the ligand 
plus the alt of R120 in the portion of the crystal that contains the ligand 
would be assigned to one occupancy group, and the alt of R120 in the portion of 
the crystal that does not contain the ligand would be assigned to the second 
group. In this case it would be appropriate to specify 'occupancy group alts 
complete', because the occupancies for the two alternative conformers of R120 
should sum to unity. Although no doubt the estimation of the occupancies would 
be dominated by the ligand in this case.

Be sure to check your B-factors after occupancy refinement to make sure the 
whole picture makes sense. Assuming your current model is essentially correct, 
from visual inspection it looks like R120 will have low occupancy and low 
B-factors when the ligand is not present (or at least B-factors consistent with 
the environment), but will have high occupancy and high B-factors when the 
ligand is present.

On another note, have you considered whether that part of the ligand could be 
modelled in a slightly different conformation, or whether there could be a 
post-translation modification?

I hope that helps,
Rob


Dr Rob Nicholls
Senior Investigator Scientist
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH



> On 5 Nov 2019, at 13:36, HK  wrote:
> 
> Dear all,
> 
> I have problem with occupancy refinement by Refmac5 for an overlapping 
> electron density of part of residue (an arginine) and part of ligand 
> (tetracyclic compound) (attached figures in Dropbox with a link as shown 
> below).
> 
> https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0
> 
> I refined part of the side chain of residue 120 and ligand (chain J residue 
> 1105) with Refmac keyword as shown below. Unfortunately, the side chain of 
> arginine moves away from the density but the ligand stays in the density. As 
> far as I understood, occupancy refinement with keyword 'occupancy group alts 
> complete' means both R120 and the ligand do not meet each other.  Did I miss 
> something from the occupancy refinement keyword?
> 
> occupancy group id 1 chain A residue 120 atom NE
> occupancy group id 1 chain A residue 120 atom CZ
> occupancy group id 1 chain A residue 120 atom NH2
> occupancy group id 1 chain A residue 120 atom NH1
> occupancy group id 1 chain A residue 120 atom CD
> occupancy group id 2 chain J residue 1105
> occupancy group alts complete 1 2
> occupancy refine
> 
> Thank you for the advice.
> 
> Best regards
> HK
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




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[ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[HK]

Dear all,

I have problem with occupancy refinement by Refmac5 for an overlapping electron 
density of part of residue (an arginine) and part of ligand (tetracyclic 
compound) (attached figures in Dropbox with a link as shown below).

https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0

I refined part of the side chain of residue 120 and ligand (chain J residue 
1105) with Refmac keyword as shown below. Unfortunately, the side chain of 
arginine moves away from the density but the ligand stays in the density. As 
far as I understood, occupancy refinement with keyword 'occupancy group alts 
complete' means both R120 and the ligand do not meet each other.  Did I miss 
something from the occupancy refinement keyword?

occupancy group id 1 chain A residue 120 atom NE
occupancy group id 1 chain A residue 120 atom CZ
occupancy group id 1 chain A residue 120 atom NH2
occupancy group id 1 chain A residue 120 atom NH1
occupancy group id 1 chain A residue 120 atom CD
occupancy group id 2 chain J residue 1105
occupancy group alts complete 1 2
occupancy refine

Thank you for the advice.

Best regards
HK



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Re: [ccp4bb] occupancy refinement

[Pavel Afonine]

Hi Mike,

Is it reasonable to refine occupancy in phenix at 2.2 A resolution?


 yes, but only for those atoms that need it.

FYI: there is a Phenix mailing list for Phenix-specific questions.

Pavel

[ccp4bb] occupancy refinement

[Michael Colaneri]

Dear all,

Is it reasonable to refine occupancy in phenix at 2.2 A resolution?

Thank you.

Mike Colaneri

Re: [ccp4bb] occupancy-refinement

[ccp4]

You are right that refining occupancy and B  value at the same time cant
be done with REFMAC, and probably wouldnt work anyway at 2.9A 

However, if you set the ligand occupancy to 0.7 say, and refine the
residue B factors, the occupancy should not be set back to 1.0. It
certainly isnt in the local version of REFMAC. 
You can judge to some extent what occupancy gives the cleanest map by
inspection. 
But at 2.9A the correlation between B and occupancy is very high and it
wilnswer. l be hard to get a definitive answer.
Eleanor
On Wed, 13 Jul 2011 10:31:59 -0500, dhurjati putcha
antharvaah...@gmail.com wrote:
 Dear CCP4ers,ncy and B value 
 
 While trying to refine a protein-ligand structure (reso=2.9A) I notice
that
 the density (2Fo-Fc)for the ligand is discontinuous. I also notice that
the
 density for the residues in the ligand binding pocket (LBP) is also very
 feeble. And when I refine the ligand occupancy the density for the
ligand
 appears and covers almost all the molecule, and the occupancy refines to
 1.0.
 
 However, I cannot refine the occupancy/B factors for the LBP residue
side
 chains because the occupancy stays at 1.0 (and if I change it manually,
it
 returns to 1) and the B factors (group  individual) remain close to the
 average for the remainder of the molecule. If I omit the LBP residue
side
 chains, I get some Fo-Fc density suggesting that these side chains are
 real.
 
 
 Is there previous evidence for such phenomena suggesting multiple
 conformations/dynamics/loose binding? Is there a way to quantify the
 dynamics associated with the side chains (as with the ligand where the
 occupancy is 1.0) so I can argue it out with potential manuscript
 reviewers? The R-factor/R free #s (22%/19%) suggest that the refinement
is
 nearing convergence.
 
 Best regards,
 
 Kumar.

[ccp4bb] occupancy-refinement

[dhurjati putcha]

Dear CCP4ers,

While trying to refine a protein-ligand structure (reso=2.9A) I notice that
the density (2Fo-Fc)for the ligand is discontinuous. I also notice that the
density for the residues in the ligand binding pocket (LBP) is also very
feeble. And when I refine the ligand occupancy the density for the ligand
appears and covers almost all the molecule, and the occupancy refines to
1.0.

However, I cannot refine the occupancy/B factors for the LBP residue side
chains because the occupancy stays at 1.0 (and if I change it manually, it
returns to 1) and the B factors (group  individual) remain close to the
average for the remainder of the molecule. If I omit the LBP residue side
chains, I get some Fo-Fc density suggesting that these side chains are real.


Is there previous evidence for such phenomena suggesting multiple
conformations/dynamics/loose binding? Is there a way to quantify the
dynamics associated with the side chains (as with the ligand where the
occupancy is 1.0) so I can argue it out with potential manuscript
reviewers? The R-factor/R free #s (22%/19%) suggest that the refinement is
nearing convergence.

Best regards,

Kumar.

[ccp4bb] Occupancy Refinement

[protein.chemist protein.chemist]

Dear All,

I have a question about the occupancy refinement of a ligand.  I have a
dataset of 2.3 angstrom and the ligand binds in multiple conformations in
the active site.
My question is if it is possible to tell which orientation(s) has/have the
highest occupancy based on occupancy refinement.
What is the best way to refine the occupancy?

Thanks,
Mariah

-- 
Mariah Jones
Department of Biochemistry
University of Florida

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Zheng Zhou]

Hi, Ed

I am dealing the similar problem. I checked CNS qindividual.inp. But how do
I refine one compound with two or more possible conformations (mainly due to
one bond rotation), each of wihich has a different occupancy? Thanks in
advance.

Joe

On Dec 17, 2007 2:24 PM, Edward Berry [EMAIL PROTECTED] wrote:

 I think the correlation between occupancy and B-factor depends
 also on the size of the ligand (relative to resolution).
 Bob Stroud, I think, has estimated occupancy by comparing
 the integrated electron density of the ligand with that of
 a well-defined, isolated water (assumed to be at unit occuancy?).

 In principle the integrated electron density is not affected
 by applying a B-factor, it is just spread out over a wider
 area. In the case of a single atom at 3 A resolution, it
 is spread out under the neighboring atoms and effectively
 lost, so it is hard to distinguish high B-factor from low
 occupancy.
 In a large ligand most of the atoms are inside the ligand,
 so their spread-out density remains inside the ligand
 and gets counted in the integrated density. In that case
 high B-factor has a very different effect than low occupancy,
 as only the latter reduces the total electron density of
 the ligand.

 During a previous reincarnation of this thread I did the
 simple test of refining occupancy and B-factor for a
 stretch of the protein (holding the rest of the protein
 at unit occupancy) in CNS 1.1, and I felt the results
 were quite satisfactory (don't have the specifics now).

 Ed

 Anastassis Perrakis wrote:
  I have already changed occupancies as Eleanor mentioned, and got
  approximate values. But my hope is to try to get much precise ones if
  possible.
 
  I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
  but in this case what you need is more accurate answers, not more
  precise ones
  (or better both, but precision alone can be a problem, and you can
  easily get
  'precise' but inaccurate data easily by making the wrong assumptions
  in your experiment)
 
  http://en.wikipedia.org/wiki/Accuracy
 
  I have heard from my colleague SHELX can refine occupancies, and
  got its license. I'll next try SHELX.
 
  I think that phenix.refine can also do occupancies ?
  The problem is not  if the program can do it, but if at your specific
 case
  you have enough information to do that in a meaningful way.
 
  For a soaking experiment and 1.5 A data, I would say that Eleanor's
  suggestion
  of tuning Occ based on B, is as close as you would get, accurate enough
  given the data,
  although not necessarily too precise.
 
  Tassos

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Edward Berry]

Zheng Zhou wrote:

Hi, Ed

I am dealing the similar problem. I checked CNS qindividual.inp. But how 
do I refine one compound with two or more possible conformations (mainly 
due to one bond rotation), each of wihich has a different occupancy? 
Thanks in advance.



Hi Zheng,
Others can answer better than I, but for what it's worth:
You have basically two methods for refining alternate,
possibly overlapping, models.
One is the alternate conformations formalism which I have
not used but is documented in the CNS FAQ
http://cns.csb.yale.edu/v1.2/faq/text.html#xtal_refine
(search for:
Q. How do I deal with alternate conformations in my refinement? )

The other way is by modeling both structures (with different chain
letter or range of residues or something) and turn off vdw
interactions between the two entities which never occur
simultaneously in the same unit cell using the igroup statement.
Some hints can be gleaned from the answer to this different question
(in the same FAQ):

Q. My structure contains a molecule which lies across a symmetry operation. This means that some parts of the molecule are mapped onto each other by symmetry. It is not a 
special position case, because no one atom lies on the symmetry operation exactly. How can I tell CNS to refine the molecule while allowing the overlap to occur?

A. You can use the igroup statement to turn the pvdw (Packing-Van-der-Waals) 
interactions off by setting the weight to zero:
  ---

Syntax for the statement is something like: interaction (selection1 selection2)
   means for it to check interaction between each atom in selection1 with
   each atom in selection2. What I am using (with CNS 1.1) is:

 igroup
   interaction ( atom_select and not (segid M or segid Z or attr store9  
0))
   ( atom_select and not (segid M or segid Z or attr store9  
0))

   interaction ( atom_select and not (segid E or attr store9  0))
   ( segid M)
   interaction ( atom_select and not (segid R or attr store9  0))
   ( segid Z)
{
   interaction ( segid E   ) ( segid M   ) weights * 1.0 pvdw 0.0 end
   interaction ( segid R   ) ( segid Z   ) weights * 1.0 pvdw 0.0 end
}   

   evaluate ($alt=1)
   while ( $alt = $nalt ) loop alcs
 interaction ( atom_select and ( attr store9 = $alt or attr store9 = 0 ))
 ( atom_select and ( attr store9 = $alt ))
 evaluate ($alt=$alt+1)
   end loop alcs
 end

Here segid M and Z are the same subunits as E and R, but in alternate 
conformations.
Store9 has to do with the formally declared alternate conformations (see atom 
selection).

The first interaction statement ignores M and Z and checks interaction of 
everything
else with everything else. The second checks interaction of everything in M with
everything except its alternate self E; the third checks Z with everything 
except R.
I see I have {commented out} the section that sets pvdw weights to zero between
alternate conformations of the same thing, but I'm not sure if this is right.
It may be this is not needed since we avoid checking those interactions, but I 
think
at one point we were turning off the NBONDS messages but not the vdw 
interaction,
so there may be two separate things needed here. I would be glad for any 
clarification.
Ed


On Dec 17, 2007 2:24 PM, Edward Berry [EMAIL PROTECTED] 
mailto:[EMAIL PROTECTED] wrote:


I think the correlation between occupancy and B-factor depends
also on the size of the ligand (relative to resolution).
Bob Stroud, I think, has estimated occupancy by comparing
the integrated electron density of the ligand with that of
a well-defined, isolated water (assumed to be at unit occuancy?).

In principle the integrated electron density is not affected
by applying a B-factor, it is just spread out over a wider
area. In the case of a single atom at 3 A resolution, it
is spread out under the neighboring atoms and effectively
lost, so it is hard to distinguish high B-factor from low
occupancy.
In a large ligand most of the atoms are inside the ligand,
so their spread-out density remains inside the ligand
and gets counted in the integrated density. In that case
high B-factor has a very different effect than low occupancy,
as only the latter reduces the total electron density of
the ligand.

During a previous reincarnation of this thread I did the
simple test of refining occupancy and B-factor for a
stretch of the protein (holding the rest of the protein
at unit occupancy) in CNS 1.1, and I felt the results
were quite satisfactory (don't have the specifics now).

Ed

Anastassis Perrakis wrote:
  I have already changed occupancies as Eleanor mentioned, and got
  approximate values. But my hope is to try to get much precise
ones if
  possible.
 
  I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
  but in this case what you need is more accurate 

[ccp4bb] Occupancy refinement of substrate using refmac5

[nkamiya]

Dear all,

This is my first mail to CCP4BB.

I'm now refining crystal structures of an enzyme at a resolution of
1.5A using refmac5 through CCP4i. Before X-ray data collections,
the substrate and co-factor ions were soaked into the crystals. Their
occupancies seem to be less than the unity because negative peaks
can be found around the substrate and co-factors in the difference
Fourier maps.

I found Developers Options in the CCP4i window of refmac5 to
specify an external keyword script file. If refmac5 can refine
occupancies of substrate and co-facotrs independently, by using the
script file, under the condition of fixed temperature factors, please
let me know examples of the script files and how to use them.

Best wishes,

Nobuo Kamiya


*
Prof. Nobuo Kamiya
email: [EMAIL PROTECTED]
fax: +81-6-6605-2557
tel: +81-6-6605-3131
Osaka City University
3-3-138, Sugimoto, Sumiyoshi, Osaka
Osaka 558-8585, Japan
*

Re: [ccp4bb] Occupancy refinement of substrate using refmac5

[Eleanor Dodson]

I dont think REFMAC can refine occupancy, but the way I would tackle it 
is to first refine B factors, then if those for the ligand are much 
higher than for the protein maybe test changing those occupancies to 0.7 
, 0,5 and so on..


But in fact B factors and occupancies are highly correlated, and you 
would probably expect a ligand soaking in after crystallisation to have 
higher B factors, so it is very difficult to get a definitive answer.

 Eleanor



nkamiya wrote:

Dear all,

This is my first mail to CCP4BB.

I'm now refining crystal structures of an enzyme at a resolution of
1.5A using refmac5 through CCP4i. Before X-ray data collections,
the substrate and co-factor ions were soaked into the crystals. Their
occupancies seem to be less than the unity because negative peaks
can be found around the substrate and co-factors in the difference
Fourier maps.

I found Developers Options in the CCP4i window of refmac5 to
specify an external keyword script file. If refmac5 can refine
occupancies of substrate and co-facotrs independently, by using the
script file, under the condition of fixed temperature factors, please
let me know examples of the script files and how to use them.

Best wishes,

Nobuo Kamiya


*
Prof. Nobuo Kamiya
email: [EMAIL PROTECTED]
fax: +81-6-6605-2557
tel: +81-6-6605-3131
Osaka City University
3-3-138, Sugimoto, Sumiyoshi, Osaka
Osaka 558-8585, Japan
*

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Anastassis Perrakis]

I have already changed occupancies as Eleanor mentioned, and got
approximate values. But my hope is to try to get much precise ones if
possible.


I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
but in this case what you need is more accurate answers, not more  
precise ones
(or better both, but precision alone can be a problem, and you can  
easily get

'precise' but inaccurate data easily by making the wrong assumptions
in your experiment)

http://en.wikipedia.org/wiki/Accuracy


I have heard from my colleague SHELX can refine occupancies, and
got its license. I'll next try SHELX.


I think that phenix.refine can also do occupancies ?
The problem is not  if the program can do it, but if at your specific  
case

you have enough information to do that in a meaningful way.

For a soaking experiment and 1.5 A data, I would say that Eleanor's  
suggestion
of tuning Occ based on B, is as close as you would get, accurate  
enough given the data,

although not necessarily too precise.

Tassos

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[George M. Sheldrick]

I thought that I would never have to disagree with both Eleanor and 
Tassos in the same email, let alone risk being burnt at a stake as a 
heretic for doubting the Gospel according to Kleywegt, but in my 
experience, given the very fortunate position that you have data to 
1.5A, the refinement of one occupancy parameter for the whole ligand 
(e.g. one SHELX free variable) will be rather well defined provided 
that sensible restraints are applied to the B-values. A common variant 
of this for ligands or side-chains is to refine one component with 
occupancy p and an alternative component or conformation with an 
occupancy 1-p, still only requiring the addition of ONE refinable 
parameter. If you are using SHELX and your ligand (or part of it) 
happens to be rigid, the rigid group refinement offers a further 
way of keeping the number of refinable parameters down. The Dundee 
PRODRG server is an excellent source of ligand geometries and 
restraints for such refinements.

George 

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry, 
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-2582


On Mon, 17 Dec 2007, Anastassis Perrakis wrote:

  I have already changed occupancies as Eleanor mentioned, and got
  approximate values. But my hope is to try to get much precise ones if
  possible.
  
 I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
 but in this case what you need is more accurate answers, not more precise ones
 (or better both, but precision alone can be a problem, and you can easily get
 'precise' but inaccurate data easily by making the wrong assumptions
 in your experiment)
 
 http://en.wikipedia.org/wiki/Accuracy
 
  I have heard from my colleague SHELX can refine occupancies, and
  got its license. I'll next try SHELX.
 
 I think that phenix.refine can also do occupancies ?
 The problem is not  if the program can do it, but if at your specific case
 you have enough information to do that in a meaningful way.
 
 For a soaking experiment and 1.5 A data, I would say that Eleanor's suggestion
 of tuning Occ based on B, is as close as you would get, accurate enough given
 the data,
 although not necessarily too precise.
 
 Tassos
 

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Santarsiero, Bernard D.]

I would also ask what is the actual goal in refining the occupancy of the
ligand?

While I agree wholeheartedly with George, the B-factors will adequately
model a lower ligand occupancy.  Usually the question you want to resolve
is Is the ligand really bound in the active site, or is this an artifact
of the model?  However, if you're trying to resolve a static disorder in
an active site, say half ligand and half solvent occupancies, then
refining the ligand occupancy can be useful.

Thus, what you really need to do is compare a refinement with just
B-factors and full occupancies vs. a refinement with the added restrained
ligand occupancy variable, in some sort of R-test, comparison of GOF, etc.
Does it really improve the model?

Bernie Santarsiero

On Mon, December 17, 2007 8:43 am, George M. Sheldrick wrote:
 I thought that I would never have to disagree with both Eleanor and
 Tassos in the same email, let alone risk being burnt at a stake as a
 heretic for doubting the Gospel according to Kleywegt, but in my
 experience, given the very fortunate position that you have data to
 1.5A, the refinement of one occupancy parameter for the whole ligand
 (e.g. one SHELX free variable) will be rather well defined provided
 that sensible restraints are applied to the B-values. A common variant
 of this for ligands or side-chains is to refine one component with
 occupancy p and an alternative component or conformation with an
 occupancy 1-p, still only requiring the addition of ONE refinable
 parameter. If you are using SHELX and your ligand (or part of it)
 happens to be rigid, the rigid group refinement offers a further
 way of keeping the number of refinable parameters down. The Dundee
 PRODRG server is an excellent source of ligand geometries and
 restraints for such refinements.

 George

 Prof. George M. Sheldrick FRS
 Dept. Structural Chemistry,
 University of Goettingen,
 Tammannstr. 4,
 D37077 Goettingen, Germany
 Tel. +49-551-39-3021 or -3068
 Fax. +49-551-39-2582


 On Mon, 17 Dec 2007, Anastassis Perrakis wrote:

  I have already changed occupancies as Eleanor mentioned, and got
  approximate values. But my hope is to try to get much precise ones if
  possible.
 
 I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
 but in this case what you need is more accurate answers, not more
 precise ones
 (or better both, but precision alone can be a problem, and you can
 easily get
 'precise' but inaccurate data easily by making the wrong assumptions
 in your experiment)

 http://en.wikipedia.org/wiki/Accuracy

  I have heard from my colleague SHELX can refine occupancies, and
  got its license. I'll next try SHELX.

 I think that phenix.refine can also do occupancies ?
 The problem is not  if the program can do it, but if at your specific
 case
 you have enough information to do that in a meaningful way.

 For a soaking experiment and 1.5 A data, I would say that Eleanor's
 suggestion
 of tuning Occ based on B, is as close as you would get, accurate enough
 given
 the data,
 although not necessarily too precise.

 Tassos

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Anastassis Perrakis]

In the danger of getting corrected by George twice in the same time  
(burning stakes excluded...)
I am not doubting at all if its doable, but if the final result will  
be more accurate / useful.


Thinking about it, with 1.5 A data it is very likely to be more  
accurate than manual handling,
but not as certain as if 1.2 A data are available. Its very likely  
that I am not up to date with resolution
limits for getting accurate results for correlated parameters as B  
and Occ and what it will really tell

you at that resolution for a bound ligand.

A.

On Dec 17, 2007, at 15:43, George M. Sheldrick wrote:



I thought that I would never have to disagree with both Eleanor and
Tassos in the same email, let alone risk being burnt at a stake as a
heretic for doubting the Gospel according to Kleywegt, but in my
experience, given the very fortunate position that you have data to
1.5A, the refinement of one occupancy parameter for the whole ligand
(e.g. one SHELX free variable) will be rather well defined provided
that sensible restraints are applied to the B-values. A common variant
of this for ligands or side-chains is to refine one component with
occupancy p and an alternative component or conformation with an
occupancy 1-p, still only requiring the addition of ONE refinable
parameter. If you are using SHELX and your ligand (or part of it)
happens to be rigid, the rigid group refinement offers a further
way of keeping the number of refinable parameters down. The Dundee
PRODRG server is an excellent source of ligand geometries and
restraints for such refinements.

George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-2582


On Mon, 17 Dec 2007, Anastassis Perrakis wrote:


I have already changed occupancies as Eleanor mentioned, and got
approximate values. But my hope is to try to get much precise  
ones if

possible.


I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
but in this case what you need is more accurate answers, not more  
precise ones
(or better both, but precision alone can be a problem, and you can  
easily get

'precise' but inaccurate data easily by making the wrong assumptions
in your experiment)

http://en.wikipedia.org/wiki/Accuracy


I have heard from my colleague SHELX can refine occupancies, and
got its license. I'll next try SHELX.


I think that phenix.refine can also do occupancies ?
The problem is not  if the program can do it, but if at your  
specific case

you have enough information to do that in a meaningful way.

For a soaking experiment and 1.5 A data, I would say that  
Eleanor's suggestion
of tuning Occ based on B, is as close as you would get, accurate  
enough given

the data,
although not necessarily too precise.

Tassos

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Edward Berry]

I think the correlation between occupancy and B-factor depends
also on the size of the ligand (relative to resolution).
Bob Stroud, I think, has estimated occupancy by comparing
the integrated electron density of the ligand with that of
a well-defined, isolated water (assumed to be at unit occuancy?).

In principle the integrated electron density is not affected
by applying a B-factor, it is just spread out over a wider
area. In the case of a single atom at 3 A resolution, it
is spread out under the neighboring atoms and effectively
lost, so it is hard to distinguish high B-factor from low
occupancy.
In a large ligand most of the atoms are inside the ligand,
so their spread-out density remains inside the ligand
and gets counted in the integrated density. In that case
high B-factor has a very different effect than low occupancy,
as only the latter reduces the total electron density of
the ligand.

During a previous reincarnation of this thread I did the
simple test of refining occupancy and B-factor for a
stretch of the protein (holding the rest of the protein
at unit occupancy) in CNS 1.1, and I felt the results
were quite satisfactory (don't have the specifics now).

Ed

Anastassis Perrakis wrote:

I have already changed occupancies as Eleanor mentioned, and got
approximate values. But my hope is to try to get much precise ones if
possible.


I never expected to preach the 'Kleywegt Gospel' in the ccp4bb,
but in this case what you need is more accurate answers, not more 
precise ones
(or better both, but precision alone can be a problem, and you can 
easily get

'precise' but inaccurate data easily by making the wrong assumptions
in your experiment)

http://en.wikipedia.org/wiki/Accuracy


I have heard from my colleague SHELX can refine occupancies, and
got its license. I'll next try SHELX.


I think that phenix.refine can also do occupancies ?
The problem is not  if the program can do it, but if at your specific case
you have enough information to do that in a meaningful way.

For a soaking experiment and 1.5 A data, I would say that Eleanor's 
suggestion
of tuning Occ based on B, is as close as you would get, accurate enough 
given the data,

although not necessarily too precise.

Tassos

Re: [ccp4bb] Fwd: [ccp4bb] Occupancy refinement of substrate using refmac5

[Gerard DVD Kleywegt]

I thought that I would never have to disagree with both Eleanor and
Tassos in the same email, let alone risk being burnt at a stake as a
heretic for doubting the Gospel according to Kleywegt, but in my


but ... but ... i haven't even said anything! i'm innocent! my name is being 
used in vain!


okay, George  Tassos, you asked for it:

--
DVD's packing list for his trip to Leeds, CCP4 meeting, January, 2008:
**

o toothpaste ... check!
o slides showing darts on a darts board to illustrate the difference
  between precision and accuracy ... check!
o one stere of vry dry wood ... check!
o matches ... check!
o 400 copies of the Gospel according to Kleywegt to be put in every
  hotel room occupied by people who attend the meeting ... check!
o autographed copy of said Gospel for each of the 10,000 members (*)
  of the DVD Fanclub on Facebook (**) ... check!
o framed picture of Mum ... errrm, no, forget that. I'm not Greek!
o one pair of clean underwear ... only if room left in suitcase
--

(*) 10,000 in binary notation
(**) http://www.facebook.com/group.php?gid=7408581677 (***)
(***) honorary chairwoman: Prof E.J. Dodson, FRS ()
() yes, really!

--dvd

(note to self: write something gospelish during the Christmas break)

**
Gerard J.  Kleywegt
[Research Fellow of the Royal  Swedish Academy of Sciences]
Dept. of Cell  Molecular Biology  University of Uppsala
Biomedical Centre  Box 596
SE-751 24 Uppsala  SWEDEN

http://xray.bmc.uu.se/gerard/  mailto:[EMAIL PROTECTED]
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**