Re: [ccp4bb] unknown density

[Barone, Matthias]

can confirm jon´s comment. I find these in virtually every high-res structure. 
some of them wobble a bit given the AA close by, such as Arg.


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, March 22, 2021 5:38:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unknown density

Definitely water pentamer, no doubt at all ;-0
Cheers, Jon.C.

Sent from ProtonMail mobile



 Original Message 
On 22 Mar 2021, 14:16, Mark J. van Raaij < mjvanra...@cnb.csic.es> wrote:

The ring looks too big to be imidazole or a nucleotide or a carbohydrate, so 
it’s probably mainly water molecules.
Perhaps partially replaced by PEG to explain the density between them (i.e. 
water molecules in most copies of the protein and PEG in some other copies). 
I’ve seen horse-shoe shaped PEG in a high-res structure before, PEGs in several 
confirmations might explain a circle.
Practically speaking, I’d first model five waters and see if they refine well.

Mark

On 22 Mar 2021, at 14:58, Sam Tang 
mailto:samtys0...@gmail.com>> wrote:

Hello fellow colleagues

Hope you are all well while the pandemics persists. I just wonder if anyone may 
have an idea what this density (looking like a pentagon) might be. The data was 
collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. Imidazole 
residual? Nucleotide (the protein itself is nucleotide-binding, but shouldn't 
be at this particular site)?

https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing

Thanks!

BRS

Sam



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Re: [ccp4bb] Characterising potential drug-binding pockets

[Barone, Matthias]

Hi Sergei

I assume that you used the term "drug-like fragments" because you would like to 
sort out candidates? The paper referenced by Bernhard is a good way to go, but 
it reflects exactly the problem of PPIs, namely that proteins with well defined 
binding pockets for substrates (such as kinases) are easily recognized while 
the algorithm has problems finding pockets of other PPIs.

I get the feeling that a more directed approach is to test the fragments in 
vitro rather than by simulation. The thought behind is to select for fragments 
that show measurable effects with fast and material-saving methods rather than 
focusing on putative pockets that might or not be recognized by your fragments.

A good example of such a work-flow is https://pubmed.ncbi.nlm.nih.gov/23344974/

best, matthias

[https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image.png]

Using a fragment-based approach to target protein-protein interactions - 
PubMed
pubmed.ncbi.nlm.nih.gov
The ability to identify inhibitors of protein-protein interactions represents a 
major challenge in modern drug discovery and in the development of tools for 
chemical biology. In recent years, fragment-based approaches have emerged as a 
new methodology in drug discovery; however, few examples of smal …




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Sergei Strelkov 

Sent: Monday, January 25, 2021 8:02:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Characterising potential drug-binding pockets


Dear everyone,


In a drug discovery project where our aim is to interfere with some PPIs, we 
could obtain binding of drug-like fragments in several potentially interesting 
pockets on our target. We would like to make a projection on how promising 
these individual pockets are. One way of doing this is through the Sitemap 
program (Halgren, T. A. Identifying and characterizing binding sites and 
assessing druggability. J. Chem. Inf. Model 49, 377–389 (2009)). Are there 
other tools around to do this? In particular, we would like to have accurate 
numbers for the pocket volume, surface, no. of H-bond donors and acceptors, 
average hydrophobicity, etc etc.


Thank you,

Sergei




Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of 
Pharmaceutical Sciences, KU Leuven O&N2, Campus Gasthuisberg, Herestraat 49 bus 
822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab 
pages: 
http://pharm.kuleuven.be/Biocrystallography



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Re: [ccp4bb] B-factors very high

[Barone, Matthias]

--Individual ADP refinement--


R-FACTORS   WEIGHT   TARGETS
 work  free  delta   data restr
28.36 32.91   4.55 14.984  101.982  0.0795.042
28.25 33.03   4.78 14.788  103.837  0.0795.023

minmax   meaniso aniso
   Overall:   47.49 211.88  96.79  10.75  2956  2930
   Protein:   47.49 211.88  96.79  10.75  2930  2930
   Water: 63.50 130.55  88.98N/A19 0
   Other:109.20 140.77 117.24N/A 7 0
   Chain  A:  74.14 153.67  99.01N/A  1474  1474
   Chain  C: 109.20 140.77 117.24N/A 7 0
   Chain  B:  47.49 211.88  94.54N/A  1456  1456
   Chain  S:  63.50 130.55  88.98N/A19 0

at this point I would def. look at the pointless and xtriage outputs...




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, January 4, 2021 4:16:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] B-factors very high

Hmm - this is extracted from your log file: it looks more sensible..


2899:  b_iso_mean   = 66.62

2907:  b_iso_min= 63.50   (limit = 1.00)

2908:  b_iso_max= 68.13   (limit = 80.00)

2909:  b_iso_mean   = 66.62


The average B is 66??


I am  more familiar with REFMAC and the fact that the iso_min and iso_max are 
so close looks odd to me, but maybe that is a PHENIX quirk?


But you need to look at the data processing log if you have access to it.

The CCP4I2 gives a very useful analysis of your data..

Eleanor



On Mon, 4 Jan 2021 at 15:03, Barone, Matthias 
mailto:bar...@fmp-berlin.de>> wrote:

dear silvia,

apart from the resolution, it would be good to know your current refinement 
strategy and if you fed the structure to pdbredo yet. How did that turn out? 
the polygon values are picked from structures at similar resolution afaik. In 
any case, a better list of B-factors is the one you find in the refinement tab 
"Atomic properties", where phenix lists them for the macromolecules and ligands 
separately. In that tab you will also find a list of which atoms cause such 
high ADPs. I think Eleanor is talking about such a screenshot

best, matthias



Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk<mailto:176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>>
Sent: Monday, January 4, 2021 3:44:33 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] B-factors very high

That polygon is not very useful I dont think. The statistics need to be given 
separately for structures solved at given resolutions.
Eleanor

On Mon, 4 Jan 2021 at 14:43, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Well - you dont give the resolution of your data or the "wilson B" which will 
be recorded in the data processing log.
If the resolution is 3A or less a) it is hard to refine a B value, and b) it 
certainly should be high..

So more information is needed.
Eleanor

On Mon, 4 Jan 2021 at 14:39, Silvia Napolitano 
mailto:silvia.napolit...@mol.biol.ethz.ch>> 
wrote:
Dear CCP4 Community,
I am currently working on the structure of a monomeric protein of 23KDa. The 
protein is generally quite "loopy" and I think I am mid-way refinement.
At the moment I am struggling, among other things, with an extremely high 
B-factor and I'm not sure how to lower it.
Do you have any suggestions on how I can proceed to obtain a lower average 
B-factor? (I am sorry if the question is rather naive, but I have worked very 
little on X-ray structures so far)
I attached a screenshot of the polygon obtained from the last refinement (the 
geometry now should be a bit improved, I am running a new job at the moment). I 
will be happy to provide further files if needed.
Many thanks in advance for your precious help!
I wish you a nice start to the new year.
Best
Silvia




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Re: [ccp4bb] B-factors very high

[Barone, Matthias]

dear silvia,

apart from the resolution, it would be good to know your current refinement 
strategy and if you fed the structure to pdbredo yet. How did that turn out? 
the polygon values are picked from structures at similar resolution afaik. In 
any case, a better list of B-factors is the one you find in the refinement tab 
"Atomic properties", where phenix lists them for the macromolecules and ligands 
separately. In that tab you will also find a list of which atoms cause such 
high ADPs. I think Eleanor is talking about such a screenshot

best, matthias



Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, January 4, 2021 3:44:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] B-factors very high

That polygon is not very useful I dont think. The statistics need to be given 
separately for structures solved at given resolutions.
Eleanor

On Mon, 4 Jan 2021 at 14:43, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Well - you dont give the resolution of your data or the "wilson B" which will 
be recorded in the data processing log.
If the resolution is 3A or less a) it is hard to refine a B value, and b) it 
certainly should be high..

So more information is needed.
Eleanor

On Mon, 4 Jan 2021 at 14:39, Silvia Napolitano 
mailto:silvia.napolit...@mol.biol.ethz.ch>> 
wrote:
Dear CCP4 Community,
I am currently working on the structure of a monomeric protein of 23KDa. The 
protein is generally quite "loopy" and I think I am mid-way refinement.
At the moment I am struggling, among other things, with an extremely high 
B-factor and I'm not sure how to lower it.
Do you have any suggestions on how I can proceed to obtain a lower average 
B-factor? (I am sorry if the question is rather naive, but I have worked very 
little on X-ray structures so far)
I attached a screenshot of the polygon obtained from the last refinement (the 
geometry now should be a bit improved, I am running a new job at the moment). I 
will be happy to provide further files if needed.
Many thanks in advance for your precious help!
I wish you a nice start to the new year.
Best
Silvia




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Re: [ccp4bb] Micro/Macro crystal seeding experience

[Barone, Matthias]

Hi Raphael

I can second Tom and Matthews comments. We got crystals only after including an 
ion exchange column (even though we work with a GST construct cleaved off with 
thrombin). Well diffracting crystals then only grow by seeding in reduced 
precipitant concentration. Additionally, the seeds allow us to test for 
additive salts in which initial crystals did not pop up. I often use a whole 
set of fine screens with different salts in which I am seeding in the protein. 
And in almost all cases, the seeding yields in good crystals only after also 
changing the protein concentration (say, from initially 12mg/ml to 8-30mg/ml 
with reduced precipitant concentration). You did not mention what you changed 
in your seeding experiment, but try a bunch of different conditions, 
concentrations (and temperature) with the seeds.

Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of hoh 

Sent: Friday, December 18, 2020 9:46:58 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Micro/Macro crystal seeding experience

Hi Raphael

Think about simple refinement,
differents kits are available

additifs kits:
  - additif, Gras and Silver bullet : from Hampton research
  - Angstrom and Morpheus from MDL
  - JBScreen Plus from Jena bioscience

detergent kits:
  - from Hampton resaerch
  - from Jena bioscience

FH



François Hoh

Centre de Biochimie Structurale,
UMR 5048 CNRS, UMR 1054 INSERM
29, rue de navacelles
34090 Montpellier Cedex, France.
Phone: +33 467 417 706
Fax:   +33 467 417 913



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Re: [ccp4bb] R free rising

[Barone, Matthias]

Hi Nika
You said you refine with phenix, correct? And that refining the phaser solution 
yielded in Rwork of 22% and a gap of 7%. For refining the rebuilt model, you 
had to open a new phenix.refine job, did you provide the native mtz there? Or 
did you maybe refine against the output mtz of the phaser refinement? I'm 
asking because you could have run the first refinent w/o Rfree flags, and upon 
choosing the output mtz, accidentally set the flags anew. That would fit to 
Dale's suggestion?



Dr. Matthias Barone
AG Kuehne, Rational Drug Design
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284

From: CCP4 bulletin board  on behalf of Wim Burmeister 

Sent: Monday, November 2, 2020 5:04:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] R free rising

...or your dataset  may have a much lower resolution than the one your initial 
model was based upon. The discrepancy between Rfree and Rwork seem to indicate 
this.
Best
Wim

De: "Eleanor Dodson" <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
À: "CCP4BB" 
Envoyé: Lundi 2 Novembre 2020 12:08:17
Objet: Re: [ccp4bb] R free rising

Yes, as Dale says when the FreeR goes up after minor rebuilding you have 
usually somehow picked up a different FreeR set..
This is almost certainly what causes this to happen - you say

This results in R free slightly lower than R work.

Small changes in a well refined structure dont change r factors very much!
Eleanor

On Mon, 2 Nov 2020 at 10:57, Dale Tronrud 
mailto:de...@daletronrud.com>> wrote:
On 11/2/2020 2:26 AM, Nika Žibrat wrote:
>
> Hello,
>
>
>
> I am trying to solve an X-ray structure of a protein of which the
> structure is already known. My aim is to only seek for ligands (soaking)
> and interpret any conformational changes. Since I am using a model with
> 100% sequence identity from PDB I am not doing Autobuild after Molecular
> phasing and continue directly with phenix.refine according to
> reccomendations (10 rounds). In accordance with X-triage I am also using
> NCS default settings in the refinement.
>
>
>
> This refinement produces solid R free and R work values around 0.29 and
> 0.22. The problem becomes when I want to manually edit the structure,
> correct the loops which are changed upon binding of the ligand, and
> correct any outliers. This results in R free slightly lower than R work.
> Upon refining, R work drops normally while R free rises significantly
> (for 0.2 -0.3). I have been trying to crack this for a few days with no
> success.
>
>
>
> I read that slightly lower R free can be normal in such cases but
> nevertheless both R values should drop, and haven’t found anything about
> the big rise of this value after refinement. It feels like I am missing
> something, since this is my first time solving a structure. Any advice?

   This is not normal behavior at all.  Rwrk and Rfree will be roughly
equal only before you perform any refinement.  The R's you report before
your model building sound quite reasonable.  When you manually change
the model you will likely cause both to increase, but you would have to
perform massive changes to get them to equalize at some larger value.

   The only thing I can think of that would cause this is for your
second refinement to be working with a newly created test set.  It is
possible that somehow you have reset your R free flags?  In an MTZ the
full data set is divided into twenty subsets -- one is the test set
while the other nineteen are the working set.  When you ran Refmac the
second time could you have told it to use a different segment as the
test set?

Dale Tronrud

>
>
>
> Thank you,
>
> Nika
>
>
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Cysteine oxidation product

[Barone, Matthias]

Hi Dhiraj

how did you measure the affinities? What are the two affinities, incl standard 
error (or even amount of data points you used in the titration exp)?

Im just asking because Im curious as what you mean by "increase". If you add 
DDT to an ITC for example, this will create a lot of background signal which 
has to be taken into account. Did you make background titrations for both?



Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Sent: Saturday, October 31, 2020 4:58:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cysteine oxidation product

Hi All
I added an extra cysteine residue to one of my protein interactions 
partners and somehow it increased the affinity in the absence of reducing agent 
however in the presence of reducing agent, the affinity is same. there is no 
disulfide bond formation between the two interaction partner as in non-reducing 
SDS-PAGE, I see the molecular weights corresponding to only the individual 
proteins and not the sum of two.
Does anyone has ever seen this? at pH 8.0, what is the probability that a 
surface exposed cysteine will have a net negative charge and still not very 
reactive? can it exist as thiolate, sulfinate or sulfonate ion? is there any 
such example in literature that anyone knows?

Thank you
Dhiraj



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Re: [ccp4bb] ligand bound to only one chain in the crystal

[Barone, Matthias]

Hi Christian
One of our proteins crystallizes always as non-crystallographic dimer. We 
occasionally find inhibitors bound to a second non-canonical site. Usually, the 
inhibitors bound to the second binding sites are sufficiently resolved only on 
one of the protein dimer. In these cases I often fail to place the second 
inhibitor at all and have to leave an unmodeled blob there. To exclude that 
this second binding site is not a crystal artifact, we used NMR HSQC to show 
that chemical shifts perturbations do occur even in solution and low inhibitor 
excess.
Best, Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Christian 
GALICIA 
Sent: Tuesday, October 27, 2020 11:19:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ligand bound to only one chain in the crystal

Hello,
In our structure only one chain in a crystallographic trimer (non-biological) 
shows a ligand bound to it (with clear density). There doesn't seem to be any 
channels (or lack of them) favoring that specific site. Can the community give 
your opinion on whether this can make the presence of the ligand or its 
biological role questionable, and give any examples of similar cases you might 
be aware of. Thank you.
--
Christian Galicia
Post Doctoral Scientist
E-mail: cgali...@vub.be





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Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?

[Barone, Matthias]

Eleanor rises a very important practical point here..."sidechains at the 
solvent interface have multiple conformations, and that as a result the water 
networks should also have partial occupancies". I was fighting with such a 
model for half a year and also tested XSHEL (there was a thread in here for 
that..). Coupling partial occupancies of sidchains with waters and other 
sidchains is a horrendously time-consuming task...and in the end, as Eleanor 
said, "correcting these details does not change the Rfactors at all". You just 
get fed up with that puzzle and stop right there.

best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Tuesday, October 20, 2020 12:40:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?

It is always hard to know when to stop tweaking a model.. We know from high 
resolution studies that many sidechains at the solvent interface have multiple 
conformations, and that as a result the water networks should also have partial 
occupancies. But usually correcting these details does not change the Rfactors 
at all - nor contribute much to the biological relevance of your structure!
So often the point to stop is when you get fed up, Phil Evans said years ago - 
I spend 95% of my time on 5% of the structure, most of which is unimportant..
In practice I let the difference maps decide when to stop - 10 Sigma peak - 
think why - lots of 5 Sigma positive and negative ones not so important
Eleanor

On Tue, 20 Oct 2020 at 11:27, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
A practice that was very popular before the Rfree came around was to fit a 
water molecule in every noise peak. One would get spectacular low Rfactors this 
way, but I cannot imagine that anyone would believe that this would be fitting 
and not over-fitting.

Best,
Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Sam Tang
Gesendet: Dienstag, 20. Oktober 2020 05:27
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] over-fitting? over-refinement?

Hi, the question may be a bit weird, but how do you define 'over-fitting' in 
the context of structure refinement? From users' perspective the practical 
aspect is to 'fit' the model into the density. So there comes this question 
from our juniors: fit is fit, how is a model over-fit?

BRS

Sam



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Re: [ccp4bb] How to save two superposed protein structures in PDB format

[Barone, Matthias]

Hi Abhik

Additional to Phils suggestions, you can use chimera to superpose and render 
the images right away.

For this, load the pdb codes right into chimera (file -> fetch ID), then tools 
-> structure comparison -> match maker

Change the Chain pairing to specific chains in ref and match structures and 
choose the according chains. After the rigid body match, just save the session 
as is and render the images.


best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Philip D. 
Jeffrey 
Sent: Monday, September 14, 2020 11:10:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to save two superposed protein structures in PDB 
format

Hello Abhik

In coot use: Calculate>Merge Molecules to append one structure to another.  
Coot will change the chain labels for you, which might work out OK in this case.

Alternatively, I've done a lot of this in my time:
Make a copy of the reference PDB file
Open it in a simple text edit (emacs, vi etc)
Go to the end, remove the PDB 'END' statement
(Many programs stop reading a PDB file when they see an END)
Append the second, superimposed file.
For most chain labels you can also change them in a text editor but 'C' 'O' and 
'N' will cause you trouble.  You can do it easily in Coot.
This would take no more than 15 seconds in emacs.

>From the command line:
grep -v 'END' reference.pdb > reference2.pdb
egrep '^ATOM|^HETATM' superimposed.pdb >> reference2.pdb
(text editor to change chain labels, if you care, but there are ways to do that 
from the command line too)

Cheers
Phil Jeffrey
Princeton


From: CCP4 bulletin board  on behalf of Abhik 
Mukhopadhyay 
Sent: Monday, September 14, 2020 4:55 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] How to save two superposed protein structures in PDB format

Hi,
How can I save two superposed protein structures in  PDB format? Is there any 
way I can do this in coot or pymol? There is only one chain in those two PDB 
structures.

Thanks in advance,
Abhik





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Re: [ccp4bb] Regarding difference in ITC and structure data

[Barone, Matthias]

Hi Monika.
Can you please give us some more information about the ITC experiments? Does 
the affinity change between maltose, maltotriose, maltotetraose and 
maltopentaose or do they all bind equally strong? And, does N stay at roughly 
equimolar ratios or is there a trend from 1 towards 0.2 for the wt protein 
(assuming your protein has one site to bind one titrand)?
I have attached a pdf to make it a bit easier to show. It could either be that 
maltose binds much weaker than the pentamer or that the pentamer binds with 1:5 
stoichiometry. In both cases you are able to get an affinity for the pentamer 
but not for the monomer, whose saturation curve would be too flat to fit. Do 
you see any dH for the maltose and maltotriose with the mutant, or is it just 
linear and not fittable like the red curve in the pdf?
The reason that you see them however in the structure could be because of 
higher protein concentration and excess. Saturation depends on both variables. 
You use 100x molar excess in the crystallization droplet, and maybe a higher 
protein concentration to easily saturate a weak interaction of, say 5-8mM. In a 
crystal you got even higher protein concentration which would allow the mutant 
to bind maltose and maltotriose even better.

So, if you see indeed some dH released by the maltose binding to your mutant, I 
would first try to push the syringe and cell concentrations to the limit. If 
you see no dH change, follow Philippe's suggestion and changing the temperature 
would be a good idea.


best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of DUMAS Philippe 
(IGBMC) 
Sent: Saturday, August 1, 2020 10:42:16 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Regarding difference in ITC and structure data

Monika
Did you try ITC experiments at different temperatures ?
Delta H may be null, or close to zero, at some temperature without implying 
that there is no binding !
Philippe Dumas


De: "monika chandravanshi" 
À: "CCP4BB" 
Envoyé: Samedi 1 Août 2020 09:57:24
Objet: [ccp4bb] Regarding difference in ITC and structure data

Dear All,
I am working on a carbohydrate-binding protein, which co-crystallizes with 
maltose, maltotriose, maltotetraose and maltopentaose and the same can be 
supported by ITC experiments as well. Also, the mutant protein (X2Y) 
co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose, 
however, the binding of only maltotriose and maltotetraose could be observed 
through ITC. For your information, the ITC conditions are the same for all the 
ligands and the ligand concentration used in ITC is same as used in 
crystallization (100x of protein concentrations). Moreover, from structural 
analysis, we have observed that the binding mode of all ligands is the same.
I request your suggestion on why maltose and maltopentaose do not show any 
binding to the mutant protein in ITC experiments.
Looking forward to suggestions.

Best Regards,
Monika



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ITC_vs_crystal.pdf
Description: ITC_vs_crystal.pdf

Re: [ccp4bb] Help With Setting up a Stereo view system

[Barone, Matthias]

Hi Marian

I used the quadro 4000 myself and had two Acer monitors connected to it, one 
with display port, one with VGA connector. Only the VGA showed 3D, the other 
would not. Have you tried going analog with this card?

best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Marian Oliva 

Sent: Thursday, July 30, 2020 2:46:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Help With Setting up a Stereo view system

Dear colleagues.

I would like to check if some one can help me with the setup of my hardware 
stereo system in Coot under Ubuntu 14

With the help of a friend I collect a bunch of second hand stuff.

1 NVIDIA 3D Video 2 system.
1 Monitor ASUS VG248 conectes through the display port
1 NVIDIA QUADRO 4000 video card.

After connecting everything and having a nightmare to install the Legacy 
Drivers 390.138

And following this instructions

https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Stereo

I run nvidia-xconfig --stereo=10

I connected the emitter both by USB and the DIN 3 pin conector
I connected the monitor through the Display Port

I reboot and , the emitter finally shows the green light

I can run nvidia-settings and I have the control panel

I see Stereo 3D Vision Stereo but only the left cube running.

But when I activate hardware stereo nothing works.

Does anybody have a clue of what to do?

Thanks in advance,

Marian


Marian Oliva
CSIC-Centro de Investigaciones Biológicas Margarita Salas
9, Ramiro de Maeztu
28040-Madrid (Spain)

phone: 0034 91 8373112 (x4380)
e-mail: mar...@cib.csic.es

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Re: [ccp4bb] protein oligomer

[Barone, Matthias]

You could try SERp? Gave some good suggestions for our non-cristallizing 
paralog. However, some of the mutants I created did not express well/were 
rather insoluble. So that might be a bad idea in your case.. anyway here's the 
link:
https://services.mbi.ucla.edu/SER/

Best, Matthias

From: CCP4 bulletin board  on behalf of 张士军 
<21620150150...@stu.xmu.edu.cn>
Sent: Monday, July 20, 2020 8:38:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein oligomer


Dear:

The protein was purified in 4 degree, and the expression level is low, so the 
aggregation is not by high concentration; the buffer pH is 7.5 which is not 
colse to the PI 8.6. It should be a dimer when function, but it was aggregated 
when negative staining. Maybe I could try to add arginine when purification, or 
do mutantions. anyone has website for prediction the mutation sites of protein?

Thanks!

Best,

shijun


-原始邮件-
发件人:"Nikolay Dobrev" 
发送时间:2020-07-19 20:29:49 (星期日)
收件人: CCP4BB@JISCMAIL.AC.UK
抄送:
主题: Re: [ccp4bb] protein oligomer


It really depends from the nature of the protein and if is 
oligomerizing/agregating/forming polymers if any of this is reversible.
On the other side if you are working with one of the fibril forming protein it 
will require optimizaiton on its on as they will form naturally polymers.

Do you observed different specises when you analyze your protein by SEC or if 
you are able to perfom DLS?
Additional information regarding your protein will be really helpful for more 
detalied suggestions how to overcome your protein.

Best,
Nikolay

Nikolay Dobrev
Scientific Officer, Protein Expression and Purification Core Facility
EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
T +49 6221 387 8633 | M +49 173 684 0532
twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of 
all EMBL events.


On 19/07/2020 14:15, S. Mohanty wrote:
Keep the protein concentration low during purification steps along with using 
other anti-aggregation agent/s. Make sure that the pH at which you are 
purifying is not close to the pI of the protein. Until completely purified, all 
purification steps should be performed in a cold room if it is a soluble 
protein.

Smita


On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga 
 wrote:


I am not sure what you mean by polymer formation. Presuming that you have 
optimized your protein concentration, pH and salt concentration, you could try 
arginine as an anti-aggregation agent in your purification (I presume you do 
FPLC). Have a look at chaotropic agents used in protein purification, The 
answer is generally dependent on the protein/proteins you are trying to purify 
and is not necessarily straightforward.

Kinds regards
[X]
On Sun, Jul 19, 2020 at 12:08 PM 张士军 
<21620150150...@stu.xmu.edu.cn> wrote:

Dear all:

Any ideas to decrease protein polymer formation? my protein was easy to form 
oligomers and precipitation when do purification,I have tried add glycerol and 
DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks!

Best Regards



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--
Nikolay Dobrev
Scientific Officer, Protein Expression and Purification Core Facility
EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
T +49 6221 387 8633 | M +49 173 684 0532
twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of 
all EMBL events.



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Re: [ccp4bb] Looking for suggestions with protein expression

[Barone, Matthias]

Hi Umar

Artem is right, we should have some more infos about how you proceed after 
disrupting the cells. The part I find curious is your usage of "usually". 
Usually soluble when? Before you centrifugate the sample? Before you go into 
the äkta? That would be a first hint to know what you mean by "usually"..

best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Umar Farook 

Sent: Saturday, June 27, 2020 6:08:51 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Looking for suggestions with protein expression

Glutathione beads for GST tagged protein.

Umar

On Sat, 27 Jun 2020, 6:56 pm John Newitt, 
mailto:newit...@gmail.com>> wrote:
On Jun 27, 2020, at 3:15 AM, Umar Farook 
mailto:umarfaroo...@gmail.com>> wrote:
>
> 
> Dear All,
>
> Sorry for an offtopic question, your suggestions are highly appreciated.
>
> We have been working on iron sulfur cluster binding protein, which is usually 
> expressed as a nice soluble protein expressed in BL21 cells but aggregated in 
> the affinity column itself and unable to recover from it. We had made n 
> number of truncations and fused to soluble tags such as MBP, but always ended 
> up in large aggregates. Anyone has experience in working with iron-sulfur 
> cluster binding protein before, please let us know the critical steps in 
> purification of such proteins, whether you have completely done the 
> expression, purification and crystallization in anaerobic conditions? or else 
> changing the expression system to eukaryotic system such as Baculo or HEK 
> 293T would help?
>
> Please share your valuable experience, thank you.
>
>
When you say “affinity column”, are you referring to a Ni2+-affinity column?
-John





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Re: [ccp4bb] Ligand building

[Barone, Matthias]

Hi Hari

If I understood you correctly, you want to modify and subsequently dock it onto 
a complex structure, right? For me, the following workflow turned out to be 
most straightfwd:

- build it in MOLOC (http://www.moloc.ch/). It lets you very easily modify an 
existing molecule without the need of a restraint file, or build a ligand from 
scratch. Adding atoms, change their H-counts, chirality, or adding bonds 
between two atoms is rather fast. Minimize the molecule either in vacuum or 
dock it right there on its substrate. If you need coot later on, store a pdb 
file and

- create the restraints file via prodrg server 
(http://prodrg1.dyndns.org/submit.html). Its up to you to disable EM at this 
point to retain the conformation of your molecule

- pre-load the cif file in coot and then load the pdb file that comes along 
with the prodrg run (prodrg might change atom names, so work with its outputs 
from now on)


If you need help in moloc, send me a message. Its not a WYSIWYG layout but has 
some big advantages over coot in terms of picking and moving single chains, 
fragments or group of atoms.

cheers,

Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Hari shankar 
<465d10db143e-dmarc-requ...@jiscmail.ac.uk>
Sent: Saturday, June 13, 2020 6:50:51 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ligand building

Hi all,

I want to build a ligand that does not exist in reality (or be able to modify 
an existing ligand as per choice).
1. JLigand in the ccp4 suite seems to work only on java and gives me an 
“configuration problem” as an error message on my Windows. I am new to this and 
unaware of how to solve this issue. Could I get some suggestions on how to 
start this?

2. Alternatively, I was trying to use eLBOW from phenix but it seems to only 
build ligands but unable to delete atoms. Is there another program or option 
for me to delete sections of the ligand and geometrical optimise them in phenix?

Thanks
Hari



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Re: [ccp4bb] Ligand Refinement in coot

[Barone, Matthias]

do you mean earlier whne coot was connected to phenix during the refinement? If 
you load the pdb and the mtz directly into coot, you have to load the cif file 
beofre starting the real space refinement in coot.


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Muhammad Bashir 
Khan 
Sent: Friday, May 1, 2020 2:25:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Ligand Refinement in coot

ReadySet

On Thu, Apr 30, 2020 at 6:23 PM Sharan Karade 
mailto:sharankar...@gmail.com>> wrote:
Hi,
 I think, you can generate new restrain file for your ligand and load before 
you do refine in COOT for next time. I have query that, how did you generate 
restrain file for ligand (elbow or readyset in phenix)?.




On Thu, Apr 30, 2020 at 5:06 PM Muhammad Bashir Khan 
mailto:m...@ualberta.ca>> wrote:
Hi Folks;

I want to ask about the ligand refinement in coot. Earlier I was able to refine 
the ligand in the coot using real space refine zone but now by some reason when 
I want to refine the ligand in coot it asks me "Refinement setup failure failed 
to find restrained for XYZ". Do not know what happen to the restrained file

All your suggestion will be highly appreciated

Bashir

--
--
Muhammad Bashir Khan, Ph.D.
Research Associate
Department of Biochemistry
Medical Science Bldg.
Lab 3-27
University of Alberta
Edmonton AB, T6G 2H7

Phone: 780-492-4577-
e-mail: m...@ualberta.ca



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--
Sharan Karade
Postdoc-fellow
IBBR-UMD, 9600 Gudelsky Dr,
Rockville
Maryland 20850




--
--
Muhammad Bashir Khan, Ph.D.
Research Associate
Department of Biochemistry
Medical Science Bldg.
Lab 3-27
University of Alberta
Edmonton AB, T6G 2H7

Phone: 780-492-4577-
e-mail: m...@ualberta.ca



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Re: [ccp4bb] superimposition of 3D structures with the DNA part only

[Barone, Matthias]

Hi Fred

Chimera let's you choose to selectively superpose chains of one entry onto a 
selective chain of the second. The easiest way is to assign the dsDNA into a 
separate chain before loading the pdb (if this is not already the case).

If you want to selectively superpose only certain atoms of one with the other 
pdb, I use a program called moloc to do a rigid body match. It's not too 
intuitive to work with but has a lot of advantages when moving and comparing 
complex structures.

Best, Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Fred. Vellieux 

Sent: Friday, April 24, 2020 1:08:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] superimposition of 3D structures with the DNA part only

Hi folks,

Some of you may have had to do this already. Either in the lab or more
recently perhaps from home.

I have two structures that I wish to superpose (two protein:dsDNA
complexes). Not using the protein part, but superposition through the
dsDNA.

I'm not quite certain what is the "best" way of doing this.

Your suggestions will be appreciated, thanks.

Fred. Vellieux

--
MedChem, 1st F. Medicine, Charles University
BIOCEV, Vestec, Czech Republic



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Re: [ccp4bb] A question of density

[Barone, Matthias]

hey Jessica

a tip that might come up later on anyway: once you put every reasonable bit 
into the desity, what I like to to when facing such blobbs: I take a well 
defined water out to create a diff density at a position where I know it is 
real. Having a feeling of how much you have to contour the diff density at that 
point can give you a good feeling how much of noise is actually in your density 
right in between the waters..?

best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Jessica Besaw 

Sent: Wednesday, March 4, 2020 6:42:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] A question of density

Hey Nukri,

Here are the details: Rwork/Rfree = 0.21 / 0.23 for a 2 Angstrom structure

I absolutely agree with you on the refinement. I did previously do that, and I 
attached the picture.

What is the BB?

Cheers!

Jessica






On Wed, 4 Mar 2020 at 12:20, Nukri Sanishvili 
mailto:sannu...@gmail.com>> wrote:
Hi Jessica,
You do not say how well is the rest of the structure refined.
First, you should refine the structure best you can, without placing anything 
in the unclear blob of your interest so to obtain the best possible phases and 
hopefully improve the blob density as well.
Then you should let the BB see what that density looks like. Looking at only 
the list of possibilities has very little value without seeing the density 
itself.
Best wishes,
Nukri

On Wed, Mar 4, 2020 at 11:10 AM Jessica Besaw 
mailto:jbesaw1...@gmail.com>> wrote:
Hello friends,

I have a "blob" of density in an active site of my protein.

I am struggling to determine if I should place a water in this spot, if I 
should model it as a disordered water, if the density may be a ligand that I 
have not considered, or if it should be left as unaccounted for density. I 
would like to publish this structure without compromising the science.

I have attached several possibilities that I have considered below.

Any suggestions would be appreciated.

Cheers!

Jessica Besaw





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Re: [ccp4bb] refinement of 0.73A data in shelxl

[Barone, Matthias]

Sorry if the mail was not clear. I figured that out now yes. As I wrote in the 
update, I found this stupid error I made and now everything looks good.

Now that I got the feeling of how shelxl works, I miss one of it's features in 
the pdb format, namely the possibility to link occupancies of a double 
confirmation to another moiety, say a water or a double confirmation of the 
ligand. It's there a way to use something similar like FVAR in a pdb file?




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: bogba...@yahoo.co.uk 
Sent: Thursday, February 6, 2020 5:01:14 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl


Hello, hope I can help.


OK, so here is the disp table...

SFAC  C H CL N O

DISP $C 0.005100.00239 15.73708

DISP $H-0.20.0  0.66954

DISP $CL0.188450.21747   1035.16450

DISP $N 0.009540.00480 28.16118

DISP $O 0.016050.00875 47.79242


If we take these coordinates...

N 30.414964   -0.1476350.11689611.00.195330.44341 =

H0A   20.427823   -0.1386560.12325611.0   -1.5

C 10.348035   -0.1607760.11097911.00.207230.28451 =

O 40.363785   -0.1741540.10290611.00.212260.22954 =

SG50.1773030.1012670.04057210.040000.068490.03024 =

O 40.2413040.0717350.03856710.960000.149820.12755 =

... the first N (followed by 3) is being assigned the scattering factors of 
chlorine because this element is 3rd in the SFAC list. The SG (followed by 5) 
is being assigned the scattering factors of O because the latter is 5th in the 
SFAC list.

I think you need to check these  assignments and the chlorine occupancy are Ok.

Jon Cooper

On 6 Feb 2020 11:13, "Barone, Matthias"  wrote:

Dear community
here is an update of my shelxl problem. I solved it after an epiphany last 
night in bed...
I tried countless things to get the postive density on the Cl under control.
Markus suggested that the density came from a radiolysed chloride, so I tried 
to superimpose chlorinated and radiolysed ligands.
However that did not lead to anything fruitful.

Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as last element:
SFAC  C  H  N  O  S  CL
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
DISP $S 0.159950.16998812.87489
DISP $CL0.188450.21747   1035.16450

The upper list only creates postive density on the Chloride, the rest of the 
map is clean and looks the same as if you would omit the DISP line of Cl 
alltogether.
The following list is coming from the .ins file of the converted prodrg file:

SFAC  C H CL N O
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $CL0.188450.21747   1035.16450
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
UNIT  38 48 1 5 7

Pasting CL as third element in the .ins file, however, created these weird 
difference signals on the backbone O and N that I mentioned.
You can probably see where this is going. Here are some atoms of the protein in 
the .ins file:

N 30.414964   -0.1476350.11689611.00.195330.44341 =
H0A   20.427823   -0.1386560.12325611.0   -1.5
C 10.348035   -0.1607760.11097911.00.207230.28451 =
O 40.363785   -0.1741540.10290611.00.212260.22954 =
SG50.1773030.1012670.04057210.040000.068490.03024 =
O 40.2413040.0717350.03856710.960000.149820.12755 =

And here are some atoms of the inhibitor:

OBM   50.3251700.4417900.18177711.00.425760.30731 = 
 <- oxygen
CE1   1   -0.0364970.2621770.18703011.00.120560.22455 = 
 <- carbon
HE1   2   -0.0288980.2473440.18766311.0   -1.2 <- proton
NAY   40.1077450.3877040.21097211.00.167190.14264 = 
<- nitrogen
CLAA   3  0.028744999  0.27121   0.199305996 0.5<- Chloride

Turned out that Jon had a good feeling about the swapping of the lines and I 
did not understand Tim's comment "The scattering factor is derived from the 
number next to the name."
Once I adjusted the numbers in the second column of my inhibitors to match the 
DISP list numbering, Rfree dropped to 16.96% and the map looks notably better 
(see attached snap shot).


Again, thank you very much for su

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Barone, Matthias]

Hi Pavel

glad you write me. I was hoping you would read my post.

- Yes, protons are added, both on the protein as well as on the molecule

- I initially only refined protein and ligand anisotropically, now Im running a 
refinement with all atoms anisotrp except Hs. This would then also be the same 
as shelxl is doing.

- Alternate conformations are modeled, also on the ligand. There are plenty, 
sure, but I think I got most of them.

- I already used Water update during refine, there are some NO3s in the 
structure. I got them in. There is a second ligand somewhere as artifact. its 
density is not well defined, so I hope to get that in once the map clears up 
more.

- I let phenix.refine optimize adp and chemisty weights, but as Petri 
suggested, Im manually increasing the scale factors to match the ones from 
shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A 
like Petri suggested and keep an eye on how tight the structure is refined in 
shelxl.


About the Rfact and the gap. Yes, thats what I was expecting. I hope if I add 
more anisotropic B fact, the Rfacts should go down to at least what shelxl 
yielded.


thank you all again for the massive feedback, ideas and help.




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: Pavel Afonine 
Sent: Monday, February 3, 2020 7:14:25 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl

Hi Matthias,

did you use correct model parameterization and optimal refinement strategy for 
the resolution? Such as:
- Add H atoms;
- Refine all but H atoms with anisotropic ADPs;
- Model alternative conformations (that one'd expect many at this resolution);
- Add solvent (water, crystallization cocktail components if you see any);
- Relax restraints on geometry and ADPs;
 long list!

If not, then what you have in terms of R factors is more or less what I'd 
expect.

In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15% 
range, and the Rfree-Rwork gap around 1-2% or less.

Since you mentioned Phenix refinement, I am happy to help you with details etc 
off-list.

Pavel

On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
mailto:bar...@fmp-berlin.de>> wrote:

Dear ccp4 community

Im having some problems solving a 0.73A structure. Spacegroup seems to be 
correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer 
shell CC1/2 24% and 90.4% complete.

The model is nearly fully built, there is no remaining unmodelled areas. 
However, Rfact is stuck 27% in phenix, with a very distinct artifact in the 
electron map (see phenix.jpg). You can see difference density on various well 
defined sidechain atoms. Notably, they seem to follow a pattern: Nearly all Val 
CG have difference signal, as well as many backbone NH. Hence, I suspected that 
it might be a problem with the SF, since we recorded the DS at 0.86A.


Hence I gave shelxl a shot:

I used the refined model from phenix, converted it via pdb2ins and pasted the 
restraints created by prodrg.

The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz 
used by phenix (no merge, friedel false).

Interestingly, shelxl can bring Rfree down to 16% and almost all of the 
diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). Except one: 
the inhibitor contains a chlorinated phenylring (pdb ligand 2L5) which now 
shows massive difference density for Cl.

I therefore suggested that I might deal with a wrong SF for Cl. Funny enough, 
pdb2ins does not produce a DISP line for Cl if converting the pdb that contains 
the inhibitor. Hence, I used pdb2ins and the pdb from PRODRG to produce SFAC 
for the inhibitor Cloride. I then pasted this line

DISP $CL0.188450.21747   1035.16450

into the .res file and updated the UNIT line. Shelxl runs through, and the 
density looks ok on the Chloride now. However Rfree is back up at 24% and the 
artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, very 
distincitvly, backbone carbonyls and NHs show difference density.

Am I right in my assumption, that the SFAC of Cloride is not properly 
calculated at the given wavelenght? And if so, how do I guess it correctly?


Thank you very much for your help!

Best, matthias




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284



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Re: [ccp4bb] nVidia 3D Vision2 glasses

[Barone, Matthias]

ah, thats good to know! I took the info from here:

https://www.nvidia.com/en-us/geforce/forums/3d-vision/41/296001/future-of-3d-vision-support-official-announcement-/

and here:

https://www.geforce.co.uk/hardware/technology/3d-vision/supported-gpus?field_gpu_type_value=All

3D Vision | Supported GPUs | 
GeForce<https://www.geforce.co.uk/hardware/technology/3d-vision/supported-gpus?field_gpu_type_value=All>
www.geforce.co.uk
Compare GeForce graphics processors that support your PC gaming system, 
including GPU performance and technical specifications.



www.nvidia.com
{{Framework.description ? Framework.description : 'Join the GeForce community. 
Browse categories, post your questions, or just chat with other members.'}}





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: Dirk Kostrewa 
Sent: Thursday, January 9, 2020 11:33:45 AM
To: Barone, Matthias; CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] nVidia 3D Vision2 glasses


Hi Matthias,


although, Nvidia announced that the final driver with 3D stereo is the 418 
driver, the more recent drivers (at least up to 440) under Linux (CentOS 7.7) 
apparently still support quad-buffered 3D stereo on our Quadro cards. This is 
in line with the 3D Vision End-of-Life 
FAQ<https://nvidia.custhelp.com/app/answers/detail/a_id/4845/~/3d-vision-end-of-life---faq>
 on the Nvidia site.


Best regards,


Dirk Kostrewa.


On 1/9/20 10:37 AM, Barone, Matthias wrote:

Hi Patricia

All modern graphics cards still support 3D vision, but you are correct that 
nvidia is addressing critical issues in release 418 only till April. So if you 
got a new card, you should not run into problems anytime soon. Just make sure 
the card is supported with the new kernel packages before updating and stick to 
the driver release 418. I did so for the last 7 years and just recently had to 
refurbish the card as it was no longer supported by centos 7.7.1908

So, I dont have an alternative yet either..

Best, matthias





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board <mailto:CCP4BB@JISCMAIL.AC.UK> 
on behalf of Patricia Borges <mailto:p.borge...@gmail.com>
Sent: Wednesday, January 8, 2020 7:25:20 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] nVidia 3D Vision2 glasses

Dear all,

Does anyone knows any alternative to the nVidia 3D Vision2 glasses for a Ubuntu 
workstation, since these are at the moment discontinued?

Thanks a lot for the help,
Best regards
Patricia



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--

**
Dr. Dirk Kostrewa
Gene Center Munich
Department of Biochemistry, AG Hopfner
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76998
E-mail: dirk.kostr...@lmu.de<mailto:dirk.kostr...@lmu.de>
kostr...@genzentrum.lmu.de<mailto:kostr...@genzentrum.lmu.de>
WWW:www.genzentrum.lmu.de<http://www.genzentrum.lmu.de>
strubio.userweb.mwn.de
**




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Re: [ccp4bb] nVidia 3D Vision2 glasses

[Barone, Matthias]

Hi Patricia

All modern graphics cards still support 3D vision, but you are correct that 
nvidia is addressing critical issues in release 418 only till April. So if you 
got a new card, you should not run into problems anytime soon. Just make sure 
the card is supported with the new kernel packages before updating and stick to 
the driver release 418. I did so for the last 7 years and just recently had to 
refurbish the card as it was no longer supported by centos 7.7.1908

So, I dont have an alternative yet either..

Best, matthias





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Patricia Borges 

Sent: Wednesday, January 8, 2020 7:25:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] nVidia 3D Vision2 glasses

Dear all,

Does anyone knows any alternative to the nVidia 3D Vision2 glasses for a Ubuntu 
workstation, since these are at the moment discontinued?

Thanks a lot for the help,
Best regards
Patricia



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Re: [ccp4bb] Potential weak binding ligand in the active site

[Barone, Matthias]

Hi Katherine

I'm regularly struggling with inhibitors that bound at non-canonical sites and 
bind there with different conformations. So the situation in these 
complex-structures could be a bit similar to your situation. In our case, the 
inhibitors bind on the non-canonical site with roughly Kd 5-20mM, causing their 
density to really wash out to an amount that parts of the molecule are no 
longer resolved due to the absence of deep binding pockets. I know the behavior 
you describe, namely that the molecule drifts over the epitope without creating 
difference density at the new position.
Sadly, I don't have a solution that would help you out for sure, but here are 
three tips:
- looking at the DS, I would assume that you have significant information 
beyond 1.9A (completeness is still 94% and CC1/2 way over 60%. How much more 
information can you bring into refinement? It took me a 1.1A DS to finally 
resolve the proper orientation of a weakly, non-canonically bound inhibitor, so 
it might be a good idea to extend the DS?
- try fem maps, might well be that they bring up important details to place the 
molecule (however, in refinement, the ligand will drift off again...)
- Use an alternate method to track down weak interactions. If I cannot resolve 
a complex satisfactorily, I switch to HSQC, which is much more sensible for Kds 
in the mM range. Just use it to confirm that the ligand is actually binding 
where you see the density (you can determine even Kds with that method)


Best,

Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Katherine Lim 

Sent: Friday, December 20, 2019 5:57:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Potential weak binding ligand in the active site

Hi all,

I apologise in advance for the long post. I am working on solving a structure 
that looks like it could have a ligand bound in the active site. My data was 
obtained from a crystal of just the soluble domain of my protein that had been 
soaked overnight in the ligand solution. The apo crystal structure is already 
known and so I have solved my structure using phaser MR. I have attached the 
Aimless report output of my structure at the end of this email. The current R 
values I have after refinement and adding in all the waters are Rfree: 0.2413 
and Rwork: 0.1897. I can clearly see green density that is much larger (only 
disappears when I contour the Fo-Fc map to about 6 A) than in my control 
crystal that had been soaked in the same concentration of just solvent (I had 
used DMF).

I am struggling to add in my ligand as it doesn't seem like the entire ligand 
can fit in the green density. We think it may be because we have only used the 
soluble domain and so the ligand isn't held very securely since the 
transmembrane domain is missing. I have been trying to fit in smaller sections 
of it and doing an occupancy refinement. So far I have been able to get part of 
the ligand in with an occupancy of about 0.6 but after the refinement run, 
phenix.refine seems to move this part of the ligand slightly out of the area 
where I had tried to fit it into the green density. Interestingly, there isn't 
a big red density in the area that this ligand section has moved to. I would 
appreciate any advice on how I should proceed with trying to figure out if I 
have tried the correct section of the ligand and the kind of refinement 
settings to use with a weak binder.

Space Group P212121
Unit cell abc   84.76, 89.84, 91.55
unit cell alpha beta gamma  90, 90, 90

OVERALL LOW RES HIGH RES
Low res limit   45.78   45.78   1.94
High res limit  1.9 9.111.9
Rmerge  0.234   0.052   1.684
Rmeas   0.244   0.054   1.768
Rpim0.069   0.016   0.527
Total # observations663073  628236851
Total # unique  55174   579 3359
I/sigma 7.5 27.91.4
CC ½0.997   0.999   0.747
Completeness %
99  98.794.7
Multiplicity12  10.811

Katherine Lim

PhD Candidate

School of Biomedical Sciences; School of Molecular Sciences

Marshall Centre for Infectious Disease Research and Training

The University of Western Australia



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Re: [ccp4bb] why does Coot ignore CONECT?

[Barone, Matthias]

Hi Pavel

I work with peptide-scaffold chimeras on a daily basis. Initially, I tried to 
include the scaffolds as HETATM and use JLigand to create the links between the 
scaffolds and the natural amino acids. This procedure however is rather fussy 
and by now I build the chimera in silico, using natural amino acids provided by 
the program. Subsequently, I run the energy minimization on my own to have a 
rough control over the conformation. I then feed the coordinates into PRODRG 
without any further minimization and use the pdb and the restraints as they 
come from the server.


Of course, such a ligand will not make use of the cif libraries of the natural 
amino acids during refinement. But if you imported the natural amino acids 
while building the chimera, your minimization used the proper restraints. I 
have the impression that this procedure is more efficient than trying to 
maintain the LINK in the pdb header (which, in my case, oftern got lost after 
running a refinement, so I had to copy-paste it anew after every cycle).


Best, Matthias



Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Pavel Mader 

Sent: Monday, November 4, 2019 6:06:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] why does Coot ignore CONECT?

Hi Paul,

thank you for your answer. My problem is that I am building a cyclic peptide 
with non-standard amino acids, the side chains of which are linked by click 
chemistry... I know how to make modified amino acids that will make peptide 
bonds with their neighbors in the polypeptide chain, but I don't know how to 
make a covalent link between side chains of amino acids say No. 3 and 12 for 
example (of a 15 AA long chain). I was trying to solve the issue by making a 
CONECT record in the pdb file, but Coot ignored it. LINK "sort of" worked, but 
it was not possible to control the geometry (bond length and angles), often the 
real space refinement simply "explodes" probably in a attempt to avoid clashes. 
My current workaround is to make a cif file for the whole 15 AA long 
polypeptide (the bonds and angles now behave more or less as expected), but I 
don't consider this as a good general practice for building long polypeptide 
chains...

Thanks in advance for any hints guiding me in the right direction.

Pavel
-- Původní e-mail --
Od: Paul Emsley 
Komu: CCP4BB@JISCMAIL.AC.UK
Datum: 1. 11. 2019 21:16:11
Předmět: Re: [ccp4bb] why does Coot ignore CONECT?
On 01/11/2019 20:17, Pavel Mader wrote:
> Hello,

Hello.

>
> I have a question, can anyone explain, why does Coot not display a covalent 
> bond manually specified by
> CONECT line in a pdb file?

Because I have never thought them necessary. Using SSBOND, LINK and residue 
dictionaries seemed to me to
cover the bases for which CONECT would be used.

Paul.



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Re: [ccp4bb] ITC question -dimer vs monomer

[Barone, Matthias]

No, the law of mass action does not depend on whether A is titrated into B or 
vice versa. The heat measured is proportional to the complex formed, relative 
to one partner being kept at constant concentration (the referece in the cell). 
The law of mass action can be re-written to describe the complex concentration 
itself as a function of A, B and Kd. Assuming a one-to-one stoichiometry, the 
function contains sums of each variable being substracted by a root function:

AB = 0.5 [ Atotal + Btotal + Kd - sqrt{  (Atotal + Btotal + Kd)^2 - 
4*Atotal*Btotal   }]

It is correct that these values should be roughly in the same range. This, 
however, is not restricted to ITC solely, but to HSQC or fluorescence-based 
titrations too. Its a direct consequence of the law of mass action.

In order to fully saturate either binding partner, say fully saturate A; AB/A 
approaching 1, all three factors need to be in the same range. The same would 
hold with B in the cell, saturating AB/B. The underlaying mass action is the 
same, just the non-ligated reference is exchanged.

For a stochiometry different from 1:1, the mass action does not yield in an 
explicit solution like the one written above. To force such a solution, one 
concentration is multiplied by a factor, N, assuming N independent, 
non-cooperative binding sites for N ligands. Lets assume a 1:2 in your case, 
2A+B2 <-> A2B2. Substituting Btotal with N*Btotal would allow to assume a 
one-to-one model with N=2. If you decide to exchange the titrant, N now 
corrects the other partner, yielding in N=0.5.





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Roger Rowlett 

Sent: Thursday, October 3, 2019 8:36:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Won't this depend on the relative final concentrations of A and B in the two 
experiments? If A going into excess B will have different mass action 
considerations that B going into excess A. Even if the final concentrations of 
A and B are stoichiometric, the initial stages of the titration will have very 
different mass action products for A into B vs. B into A. An additional wrinkle 
is the concentrations of A and B relative to the dissociation constant Kd. The 
titration curve math gets a little more complex when the concentration of the 
species is in the same order of magnitude as the Kd. There are quite a few 
examples of bollixed binding curves in the literature for tight-binding 
equilibria that ignore the relationship between Kd and ligand concentrations.  
Cooperativity issues will of course perturb any pure, non-cooperative 
statistical analysis based on equilibrium constants.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu

On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp 
mailto:hofkristall...@gmail.com>> wrote:
I am not looking for anything yet – I wonder what – if any – the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don’t understand what you are trying to do—are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dime

Re: [ccp4bb] ITC question -dimer vs monomer

[Barone, Matthias]

As Reza already pointed out, ITC cannot tell you anything about the 
sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> 
AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a 
fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2.

Given the lack of additional information, its probably good to assume a simple 
one-to-one model and titrate either partner to the other.

If you have two separate binding steps (with similar Kd for B2 for A as well as 
AB2 for A), you would measure an apparent affinity and would see the 
stochiometrics according to  the inflection point (be it around equimolar 
excess or at 0.5 or 2, depending on whether you titrate A or B). If the 
reaction is more complicated and the the affinities for B2 for A differ 
significantly much from the affinity of AB2 for A, then a simple one-to-one 
would leave some notable information in the residual standard deviations 
(meaning, the residuals would not spread normally around the Regression line, 
but should show a wavy pattern).

Sorry for the long mail..

Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Michael Fairhead 

Sent: Thursday, October 3, 2019 5:59:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Hello,
If you Google Alan cooper ITC insulin, you should find his work describing the 
study of insulin dimer:monomer equilibrium and the effect of cyclodextrin 
studied via ITC. This may be of some help.
Cheers
Mike

https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf&ved=2ahUKEwiOpoeisYDlAhUDThUIHX5mAyMQFjAAegQIAhAB&usg=AOvVaw0Ng-W03upy4DG12unFGDY3


From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet – I wonder what – if any – the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don’t understand what you are trying to do—are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--

Re: [ccp4bb] challenges in structural biology

[Barone, Matthias]

Something that pops up here routinely, connected to 5) what does "resolution" 
really mean?:

While new methods are being implemented to improve the accuracy of models or to 
deal with weak data, how to convince editors to accept the new methods?

cheers, matthias





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Frank Von Delft 

Sent: Tuesday, July 16, 2019 6:34:16 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] challenges in structural biology

1.1  Getting many diverse conformations routinely into well-diffracting
crystals;  and knowing how to interpret them biologically.

On 15/07/2019 20:44, Holton, James M wrote:
> Hello folks,
>
> I have the distinct honor of chairing the next Gordon Research
> Conference on Diffraction Methods in Structural Biology (July 26-31
> 2020).  This meeting will focus on the biggest challenges currently
> faced by structural biologists, and I mean actual real-world
> challenges.  As much as possible, these challenges will take the form of
> friendly competitions with defined parameters, data, a scoring system,
> and "winners", to be established along with other unpublished results
> only at the meeting, as is tradition at GRCs.
>
> But what are the principle challenges in biological structure
> determination today?  I of course have my own ideas, but I feel like I'm
> forgetting something.  Obvious choices are:
> 1) getting crystals to diffract better
> 2) building models into low-resolution maps (after failing at #1)
> 3) telling if a ligand is really there or not
> 4) the phase problem (dealing with weak signal, twinning and
> pseudotranslation)
> 5) what does "resolution" really mean?
> 6) why are macromolecular R factors so much higher than small-molecule ones?
> 7) what is the best way to process serial crystallography data?
> 8) how should one deal with non-isomorphism in multi-crystal methods?
> 9) what is the "structure" of something that won't sit still?
>
> What am I missing?  Is industry facing different problems than
> academics?  Are there specific challenges facing electron-based
> techniques?  If so, could the combined strength of all the world's
> methods developers solve them?  I'm interested in hearing the voice of
> this community.  On or off-list is fine.
>
> -James Holton
> MAD Scientist
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1





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Re: [ccp4bb] AKTA FPLC fractionation peak by peak

[Barone, Matthias]

Hi sorin
The problem in your elution profile is that you cannot define a global 
threshold as the baseline is heavily bent (relative to the peak heights) and 
starting to fractionate at 6' is not the problem, telling the script to stop 
once you reach baseline at 13' is.
I would try what you proposed: Given that your peak elution time points are 
reproducible, I'd actually work with the "watch" function and turn it on while 
watching OD for the given time windows.  fractionate the first block when OD 
exceeded a certain threshold and then turn the watch function off after, say 
13'. Turn it back on at 20' and fractionate into another well. Use the time as 
base and keep the Äkta pumping at constant speed.
Curious if someone knows a better strategy!
Best, matthias


From: CCP4 bulletin board  on behalf of Sorin Draga 

Sent: Monday, June 3, 2019 8:29:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AKTA FPLC fractionation peak by peak

Dear all,

We have an old akta system, running Unicorn 4.0, recently donated to our lab. 
While attempting to fractionate a protein mixture, I was unable to convince 
Unicorn to fractionate within given limits (say, for ex peak A between RT a and 
b and peak B between retention times c and d). All that I could manage to do is 
fractionate at a fixed time interval.
For clarity, I have attached a picture below (orange square would be what we 
actually want to fractionate)
Any help would be appreciated, as we are pressed for time ( I am certain that 
we are missing something simple)

Thank you!
[image.png]




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Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang)

[Barone, Matthias]

I agree with Pavel... if you got so many observations you dont need TLS but use 
anisotropic adps. What I want to add is the dangerous comment to "cutting back 
data ... to improve Rfact" Of course, if you lower the amount of observations 
while keeping the parameters to fit constant will lower your R facts. That 
doesnt necessarily mean you improve your model. It just means that you incase 
the param to obs ratio - which says nothing about the accuracy of your model...


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Pavel Afonine 

Sent: Wednesday, October 10, 2018 11:03:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution 
dataset (1.05 Ang)

Hi Tony,


I always feel some people are too "greedy" with the resolution they want to 
achieve. I mostly find that extremely high density is a pain to work with as 
it's usually accompanied by many dual, triple conformers, a lot of noise in the 
solvent phase that is often difficult to interpret, etc... Often you spend more 
time on a high resolution structure that clearly shows your protein, as opposed 
to a low resolution structure where it's difficult to interpret parts of the 
map. Also, in most cases you REALLY don't need a 1 Ang map to clearly show the 
overall structure of your protein, ligands, first shell of solvent molecules on 
the surface of your protein, etc...

higher resolution typically means you have a chance to obtain a more accurate 
atomic model of a crystal structure, which in turn may lead to a more accurate 
interpretation of this model. I fully agree that high resolution requires more 
modeling effort. There is still a great room for software developers to provide 
more automation.


Your completeness is 90% in your high resolution shell, which is fine, but have 
you checked you can clearly see most reflections for h, k and l? Maybe you're 
missing many reflections for one of them. I would at least try cutting your 
data back to 1.1 or 1.2 Ang, as it might dramatically improve your R factors 
and still show everything you want to show.


Also, did you try TLS refinement?

At resolutions like 1.2A or better one normally models ADPs as anisotropic for 
all atoms (except H), so no need to use TLS.

Pavel




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Re: [ccp4bb] Creating map volume of custom size

[Barone, Matthias]

I use CCP4 mapmask to exted the map some Å beyond the

asymmetric unit in order to see the density at crystal contacts




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Phil Evans 

Sent: Friday, July 13, 2018 12:14:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Creating map volume of custom size

I think the CCP4 program map rot will do this
Phil

> On 13 Jul 2018, at 10:16, melanie.voll...@diamond.ac.uk 
>  wrote:
>
> Hello everyone,
>
>
> I have a question about manipulating map files.
>
>
> I want to use my map derived from X-ray diffraction and create a standard 
> volume for it, say 300A3.
>
> This is to be able to create slices of equal dimensions along all three 
> directions of the electron density stored in the map file. Currently my 
> slices differ in dimensions as the default volume is defined by the unit cell.
>
>
> I don't think there is anything in the map tools from the X-ray field I know. 
> I had a play with Chimera and I think there is a tool in there to use some 
> form of reference cell created from coordinates. But I don't want to make use 
> of a coordinates file.
>
>
> I just want to inflate my unit cell to a standard volume.
>
>
> Any ideas would be very welcome.
>
>
> Thanks
>
>
> Melanie
>
> --
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Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Barone, Matthias]

we also do structure-activity relationship and rational drug design. And I 
agree with Christian: never rely on one single method and try to include a 
homogenous assay, such as ITC or FT. you mention a tyrosine involved in the 
binding pocket. Did you try to track the Tyr in FT?

best, matthias


From: CCP4 bulletin board  on behalf of Christian Roth 

Sent: Friday, April 27, 2018 9:00:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Compound with flexible conformation but nM Kd

Anectodal evidence I have heard from colleagues working with things, which are 
immobilized is that the measured Kd value on the surface can be wildly 
different from what is measured in solution. A superbinder on a surface might 
not be as good in solution. There seems still a lot of debate why that is.

Cheers
Christian

On Fri, Apr 27, 2018 at 5:07 AM, WENHE ZHONG 
mailto:wenhezhong.xmu@gmail.com>> wrote:
Hi Philippe,

The affinity was measured by SPR where we immobilized the protein on the chip. 
One thing I forgot to mention is that the association rate (kon) shown in SPR 
experiment for this compound is faster (>10-fold faster) compared to other 
analogues with similar koff. There is a pi-pi interaction between the scaffold 
structure and the protein (tyrosine ring). Is it possible that the hydrophobic 
substituent could facilitate the formation of this pi-pi interaction but not 
necessary to involve in the interaction? Thanks.

Kind regards,
Wenhe

On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) 
mailto:p.du...@ibmc-cnrs.unistra.fr>> wrote:


Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG 
mailto:wenhezhong.xmu@gmail.com>> a écrit:

Just to be sure: how was the nM affinity evaluated ? By in vitro measurements, 
or by obtaining an IC50 by tests on cells ?
Of course, if you are mentioning an IC50, you may have a measurement of the 
efficacy of drug entrance in the cells, not just of specific binding to your 
protein target.
Philippe D.

Dear Community,

A little bit out of topic here. We are applying the structure-based approach to 
design compounds that can bind our protein target. We have synthesized a series 
of analogues based on the same scaffold with different substituents at one 
particular site. The most potent analogue (nM Kd) has a long alkyl chain 
substituent. We thought this hydrophobic substituent should have strong 
interactions with the target protein leading to nM range affinity. However, 
crystal structures show very weak densities for this substituent and no obvious 
interaction between the substituent and the target protein, suggesting that 
this long alkyl chain substituent is flexible without binding to the protein. 
This binding site is relatively negative charged according to the electrostatic 
potential analysis.

So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
substituent can lead the compound to achieve nM affinity (>10-fold better than 
any other substituent) — in particular the binding site is not hydrophobic and 
no interaction is found between the substituent and the protein.

Anything I have miss here that can increase the binding affinity without 
interacting with the target?

Thanks.

Kind regards,
Wenhe