Re: [ccp4bb] A question about Cys and fluoro benzene ring

[CRAIG A BINGMAN]

As others have suggested, it looks very much like a substitution at the 
fluorine position.  I might add that high resolution electrospray mass spec 
should be very useful in this case, because I suspect that this adduct will  
survive sample preparation, and it would provide strong and orthogonal 
experimental evidence that a chemical reaction had occurred at that position, 
specifically that fluorine had been lost during the reaction. To make your life 
easier, I’d definitely run an unreacted protein sample as a control (subjecting 
it to the full experimental procedure for creating the reaction.) The mass 
resolution of electrospray is amazing, but in many cases there are 
complications to the spectrum from associated ions. You definitely want the 
control spectrum for the unreacted protein to be from exactly the same solution 
conditions as your experimental sample.

> On Aug 23, 2017, at 10:01 AM, Cheng Zhang  wrote:
> 
> Hi everyone,
> 
> We recently got a structure of a receptor bound to a ligand. The ligand has a 
> fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
> receptor. The density for the ligand and the Cys seems to suggest a covalent 
> bond. However, I don't know if a covalent bond is chemically possible. Also, 
> I believe Cys is rarely involved in cation-pi interactions? Any suggestions 
> for placing the Cys and the fluoro methyl benzene ring?
> 
> Thanks! 
> 
> Cheng
> 
> 
> 
> -- 
> -
> Cheng Zhang

Re: [ccp4bb] AW: [ccp4bb] Unknown positive electron density

[Betty Chu]

Thank you all for your replies and suggestions.

I have fitted in some dummy waters as Herman suggested and this is the
result after one round of refinement. The waters are positioned where there
is highest electron density, but I am not sure how to move forward from
here.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/VTQ6Uinp1UVIWGR
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/NUZKClayzSMn7PP
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/0FZhxtgcQhhqnaA

I have also tried fitting in a nucleotide, but the geometry of it does not
fit the density well. We do not think this is due to Fourier truncation
ripples since the positive density is not at the crystallographic symmetry
axis.

Betty

On Tue, Aug 22, 2017 at 10:34 AM, Pradeep Pallan 
wrote:

> Hi Betty,
> Could you post a few more screen shots of different orientations of the
> electron density?
>
> If you see waters that are 1.6, 1.9 and 2.2 A away from the metal ion, I
> would try
> Mg2+ ion (trace amount of metal ions could be present in salt
> solutions/buffers as an impurity)
> and refine it (although 1.6Å is too close). In general, Mg2+ ions would
> show an octahedral
> geometry.
>
>
>
>
>
> On Tue, Aug 22, 2017 at 3:35 AM,  wrote:
>
>> Dear Betty,
>>
>>
>>
>> You have very high resolution, which helps you to identify your ligand,
>> but the ligand may be disordered…
>>
>> What I would do is to place some dummy atoms (e.g. waters) and refine and
>> look if the molecule gets clearer. By scrolling the density in coot, you
>> can identify the positions with the highest electron density were you
>> should put your dummy atoms.
>>
>>
>>
>> Looking at your pictures, as far as is possible without the ability to
>> rotate them, I would try to fit deoxyribose-phosphate, or even a complete
>> nucleotide. Maybe your prep contained some unreacted nucleotides and your
>> anomalous peak is phosphorus.
>>
>>
>>
>> Good luck!
>>
>> Herman
>>
>>
>>
>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
>> von *Betty Chu
>> *Gesendet:* Montag, 21. August 2017 17:19
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [ccp4bb] Unknown positive electron density
>>
>>
>>
>> Dear ccp4bb,
>>
>> I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While
>> the model for the DNA fits very well into the density, there is a patch of
>> positive electron density in the solvent space that we are having trouble
>> with.
>>
>> The screenshot can be viewed through this link:
>> https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC
>> 
>>
>> In the screenshot, the yellow color is the anomalous map and a barium ion
>> is fitted into density near the positive green electron density.
>>
>>
>> The oligonucleotide was purchased from IDT. The crystallization condition
>> is 15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling
>> Ba2+ with coordinated waters, MPD, and sodium cacodylate into the electron
>> density, but none of those fit well.
>>
>> Any suggestions regarding the identity of this electron density is much
>> appreciated. Thank you!
>>
>>
>>
>> Sincerely,
>>
>> Betty Chu
>>
>> Paukstelis Research Group
>>
>> Department of Chemistry and Biochemistry
>>
>> University of Maryland, College Park
>>
>
>
>
> --
>
> ---
> Pradeep Pallan
>

[ccp4bb] Job opportunity

[Brian Mark]

Hello everyone,

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==
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[ccp4bb] AW: [ccp4bb] A question about Cys and fluoro benzene ring

[Herman . Schreuder]

Dear Cheng,

You could check whether you get irreversible inhibition with your compound, 
which could be a sign of a covalent link with the protein.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Savvas 
Savvides
Gesendet: Mittwoch, 23. August 2017 21:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] A question about Cys and fluoro benzene ring

Dear Cheng,

it is possible that the sulfhydryl of the cysteine might have reacted with the 
methyl fluoro benzyl group via nucleophilic aromatic substitution. Fluorine is 
not the best leaving group among the halides but it would certainly allow this 
especially if the ligand-protein incubation time was sufficient and if the the 
pH of the reaction is above 7. This would get the Cys-SH closer to the 
cysteinyl moiety (S:-). The pKa of the Cys-SH group is just under 8.5 and in 
your case it might even be lower depending on local structure environment, 
resulting in an even better nucleophile.

Your electron density indeed suggests that the sulfur from the Cys-SH is 
covalently bonded to the benzyl ring and this would happen at the carbon that 
originally carried the fluorine. You will have to remodel your ligand, keeping 
in mind that the fluorine will no longer be part of your ligand model. The 
methyl group will of course be retained.

It might also be useful to try to confirm the reactivity of the ligand of 
interest with the protein (reminiscent of a suicide or covalent inhibitor 
against cysteine proteases?) via some kind of an orthogonal method (e.g. mass 
spectrometry). This would even tell you if the particular cysteine in your 
structure is truly the relevant covalent target. In this way you can even 
compare washed and dissolved crystals of your protein-ligand complex with the 
complex in solution.

best wishes
Savvas


---
Savvas Savvides
VIB-UGent Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx




On 23 Aug 2017, at 17:01, Cheng Zhang 
> wrote:

Hi everyone,

We recently got a structure of a receptor bound to a ligand. The ligand has a 
fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
receptor. The density for the ligand and the Cys seems to suggest a covalent 
bond. However, I don't know if a covalent bond is chemically possible. Also, I 
believe Cys is rarely involved in cation-pi interactions? Any suggestions for 
placing the Cys and the fluoro methyl benzene ring?

Thanks!

Cheng



--
-
Cheng Zhang

Re: [ccp4bb] refmac-extra-params

[Paul Emsley]

On 24/08/17 16:29, Dr A.A. Jalan wrote:


I am trying to change the default refmac run parameters in coot. For 
this, I created a refmac-extra-params file in the directory from where 
coot is launched.


(set! refmac-extra-params (list "MAKE HYDROGENS ALL" "REFI BREF ANIS"))



You can set refmac-extra-params in 2 ways. Either add to your ~/.coot file:

(set! refmac-extra-params (list "MAKE HYDROGENS ALL" "REFI BREF ANISO"))

or recreate a file called refmac-extra-params (like you have done) with 
contents


MAKE HYDROGENS ALL
REFI BREF ANISO

Sorry if that was not clear in the documentation.

Regards,

Paul.

[ccp4bb] refmac-extra-params

[Dr A.A. Jalan]

 

Dear all, 

I am trying to change the default refmac run parameters in coot. For
this, I created a refmac-extra-params file in the director from where
coot is launched. 

(set! refmac-extra-params (list "MAKE HYDROGENS ALL" "REFI BREF ANIS")) 

But these modification are ignored through the following message in the
log file, 

Data line--- MAKE HYDROGENS NO
 Data line--- NCYCLES 0
 Data line--- WEIGHT AUTO 5
 Data line--- (set! refmac-extra-params (list "MAKE HYDROGENS ALL" "REFI
BREF ANIS"))
===> Warning: Unrecognized keyword: (SET
 Data line--- END 

I was wondering if I am missing something. I have also tried "set"
without the "!" and "define" but in vain. 

Please could anyone point me to a solution. 

best regards, 

Abhishek 
 

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[Ardan Patwardhan]

Dear All

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Both positions are based at the European Bioinformatics Institute (EMBL-EBI), 
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Best Wishes

Team Leader - Cellular Structure & 3D Bioimaging
EMDB & EMPIAR
European Bioinformatics Institute (EMBL-EBI) European Molecular Biology 
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Wellcome Trust Genome Campus
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[ccp4bb] Reposting with link included - Postdoctoral position at Birkbeck

[Gabriel Waksman]

Link now included - apologies

Postdoctoral Researcher – Cryo-Electron Microscopy and Cell Biology of Type IV 
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The ISMB at Birkbeck is seeking one Post-doctoral Research Assistant to carry 
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