Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Jan Dohnalek]

We have seen quite a few of these.
Oxidation of S.
Mostly in our case a result if reaction with beta-mercaptoethanol..

Jan


On Mon, Dec 18, 2023 at 6:14 PM Liliana Margent <
lmarg...@gradcenter.cuny.edu> wrote:

> Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc
> density from a few cysteines during the refinement process. We were
> wondering if anyone had seen something similar so they could offer some
> insight on the likely chemistry at hand, and a potential refinement
> solution. Attached are two images of the observed extra density at two
> cysteines, 505 and 518. We have modeled acetylated cysteine,
> s-hydroxycysteine, and s-mercaptocysteine but it does not solve the
> density. The protein in question is a Protein Tyrosine Phosphatase known as
> STEP (PTPN5), with data collected to a resolution of 1.79 Å. The crystals
> were grown in bis-tris pH 6.65, 200mM Li2SO4, ~30% PEG3350. Of note, prior
> to data collection the crystal was conserved at room temp for long time
> where it dried, and was subsequently rehydrated with mother liquor. Thank
> you so much.
> --
>
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-- 
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
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252 50 Vestec near Prague
Czech Republic

Tel. +420 325 873 758



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Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Jon Cooper]

Hello, you seem to have positive difference density for the Cys sulphur/sulfur 
atom. It would be worth checking it's occupancy.

Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile

 Original Message 
On 18 Dec 2023, 17:03, Liliana Margent wrote:

> Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc 
> density from a few cysteines during the refinement process. We were wondering 
> if anyone had seen something similar so they could offer some insight on the 
> likely chemistry at hand, and a potential refinement solution. Attached are 
> two images of the observed extra density at two cysteines, 505 and 518. We 
> have modeled acetylated cysteine, s-hydroxycysteine, and s-mercaptocysteine 
> but it does not solve the density. The protein in question is a Protein 
> Tyrosine Phosphatase known as STEP (PTPN5), with data collected to a 
> resolution of 1.79 Å. The crystals were grown in bis-tris pH 6.65, 200mM 
> Li2SO4, ~30% PEG3350. Of note, prior to data collection the crystal was 
> conserved at room temp for long time where it dried, and was subsequently 
> rehydrated with mother liquor. Thank you so much.
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1



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Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Wim Burmeister]

Hello, 
this looks like beta-mercaptoethanol adduct through a S-S bond. 
See 
[ https://pubmed-ncbi-nlm-nih-gov.insb.bib.cnrs.fr/12421561/ | The crystal 
structure of the Epstein-Barr virus protease shows rearrangement of the 
processed C terminus.  ] 
Buisson M, Hernandez JF, Lascoux D, Schoehn G, Forest E, Arlaud G, Seigneurin 
JM, Ruigrok RW, Burmeister WP. J Mol Biol. 2002 Nov 15;324(1):89-103. doi: 
10.1016/s0022-2836(02)01040-9. 

Wim 

De: "Liliana Margent"  
À: "CCP4BB"  
Envoyé: Lundi 18 Décembre 2023 18:03:46 
Objet: [ccp4bb] extra Fo-Fc density in two Cysteines 

Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc 
density from a few cysteines during the refinement process. We were wondering 
if anyone had seen something similar so they could offer some insight on the 
likely chemistry at hand, and a potential refinement solution. Attached are two 
images of the observed extra density at two cysteines, 505 and 518. We have 
modeled acetylated cysteine, s-hydroxycysteine, and s-mercaptocysteine but it 
does not solve the density. The protein in question is a Protein Tyrosine 
Phosphatase known as STEP (PTPN5), with data collected to a resolution of 1.79 
Å. The crystals were grown in bis-tris pH 6.65, 200mM Li2SO4, ~30% PEG3350. Of 
note, prior to data collection the crystal was conserved at room temp for long 
time where it dried, and was subsequently rehydrated with mother liquor. Thank 
you so much. 



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 | website ] [ 
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Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Roberto Steiner]

Difficult to be sure from a single angle pic but I would suggest Cys oxidation.

There is an important 20 years old paper (see below) on Cys oxidation in a 
tyrosine phosphatase (like yours)

Best
Roberti]


Nature

  *   

  *   

  *   

. 2003 Jun 12;423(6941):773-7.
 doi: 10.1038/nature01681.
Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B
Rob L M van 
Montfort
 1, Miles 
Congreve, 
Dominic Tisi, 
Robin Carr, 
Harren Jhoti


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
Viale G. Colombo 3
35131 Padova, Italia
Telefono 0039 049 8276409

Responses to emails are not expected outside of your normal working hours.








On 18 Dec 2023, at 18:03, Liliana Margent 
mailto:lmarg...@gradcenter.cuny.edu>> wrote:

You don't often get email from 
lmarg...@gradcenter.cuny.edu. Learn why 
this is important
Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc 
density from a few cysteines during the refinement process. We were wondering 
if anyone had seen something similar so they could offer some insight on the 
likely chemistry at hand, and a potential refinement solution. Attached are two 
images of the observed extra density at two cysteines, 505 and 518. We have 
modeled acetylated cysteine, s-hydroxycysteine, and s-mercaptocysteine but it 
does not solve the density. The protein in question is a Protein Tyrosine 
Phosphatase known as STEP (PTPN5), with data collected to a resolution of 1.79 
Å. The crystals were grown in bis-tris pH 6.65, 200mM Li2SO4, ~30% PEG3350. Of 
note, prior to data collection the crystal was conserved at room temp for long 
time where it dried, and was subsequently rehydrated with mother liquor. Thank 
you so much.


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Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Parthasarathy Sampathkumar]

Hi Liliana,
May be it would be good to know if this adduct was formed during
crystallization or protein was modified prior to crystallization.  One
could consider performing a protease digestion, followed by LC-MS/MS to
determine the molecular weight of the adduct and then work backwards to
account for the difference in dalton.

Good Luck,
Partha

On Mon, Dec 18, 2023 at 9:14 AM Liliana Margent <
lmarg...@gradcenter.cuny.edu> wrote:

> Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc
> density from a few cysteines during the refinement process. We were
> wondering if anyone had seen something similar so they could offer some
> insight on the likely chemistry at hand, and a potential refinement
> solution. Attached are two images of the observed extra density at two
> cysteines, 505 and 518. We have modeled acetylated cysteine,
> s-hydroxycysteine, and s-mercaptocysteine but it does not solve the
> density. The protein in question is a Protein Tyrosine Phosphatase known as
> STEP (PTPN5), with data collected to a resolution of 1.79 Å. The crystals
> were grown in bis-tris pH 6.65, 200mM Li2SO4, ~30% PEG3350. Of note, prior
> to data collection the crystal was conserved at room temp for long time
> where it dried, and was subsequently rehydrated with mother liquor. Thank
> you so much.
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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