Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Jonathan Cooper 
 Sorry, that was the wrong zenodo link! The correct one is: 
https://doi.org/10.5281/zenodo.220983
On Friday, 27 March 2020, 23:06:01 GMT, Jonathan Cooper 
<0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote:  
 
  If you can model it as a lysine, it will be the beta-ME adduct. There's a 
good one in 1b4e (see attached) which Eileen Jaffe pointed out to me and I 
never got round to sorting out. Now there's another one of 'mine' for which the 
data have not been deposited, but I did put the images on zenodo 4 years ago 
(https://doi.org/10.5281/zenodo.51560) if anyone fancies a little lock-down 
project!!


On Friday, 27 March 2020, 22:02:20 GMT, CRAIG A BINGMAN 
<21371e2fba31-dmarc-requ...@jiscmail.ac.uk> wrote:  
 
 My guess would be that the chains were prepared under reducing conditions, 
mixed, and then allowed to oxidize. The surface adduct could easily form during 
this process.


On Mar 27, 2020, at 4:38 PM, Cowan, Richard H. (Dr.)  
wrote:
Hi,
The Fab constructs have a c-terminal cysteines on both the heavy and light 
chains, which should form a disulphide. Adding reducing agent to the 
purification of the protein would potentially reduce this disulphide, possible 
causing issues the stability and heterogeneity? At least that's my 
understanding? 

Thanks,

Dr Richard Cowan
Research Associate
 HWLSB 1/05Department of BiochemistryUniversity of LeicesterLancaster 
RoadLeicester, LE1 9HN, U.K. Phone +44 (0) 116 229 7077
From: CCP4 bulletin board  on behalf of Paul Emsley 

Sent: 27 March 2020 21:33
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct 
On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:



  although [BME] seems unlikely, since the crystallized protein is a Fab.



I don't follow.

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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Jonathan Cooper 
 If you can model it as a lysine, it will be the beta-ME adduct. There's a good 
one in 1b4e (see attached) which Eileen Jaffe pointed out to me and I never got 
round to sorting out. Now there's another one of 'mine' for which the data have 
not been deposited, but I did put the images on zenodo 4 years ago 
(https://doi.org/10.5281/zenodo.51560) if anyone fancies a little lock-down 
project!!


On Friday, 27 March 2020, 22:02:20 GMT, CRAIG A BINGMAN 
<21371e2fba31-dmarc-requ...@jiscmail.ac.uk> wrote:  
 
 My guess would be that the chains were prepared under reducing conditions, 
mixed, and then allowed to oxidize. The surface adduct could easily form during 
this process.


On Mar 27, 2020, at 4:38 PM, Cowan, Richard H. (Dr.)  
wrote:
Hi,
The Fab constructs have a c-terminal cysteines on both the heavy and light 
chains, which should form a disulphide. Adding reducing agent to the 
purification of the protein would potentially reduce this disulphide, possible 
causing issues the stability and heterogeneity? At least that's my 
understanding? 

Thanks,

Dr Richard Cowan
Research Associate
 HWLSB 1/05Department of BiochemistryUniversity of LeicesterLancaster 
RoadLeicester, LE1 9HN, U.K. Phone +44 (0) 116 229 7077
From: CCP4 bulletin board  on behalf of Paul Emsley 

Sent: 27 March 2020 21:33
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct 
On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:



  although [BME] seems unlikely, since the crystallized protein is a Fab.



I don't follow.

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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread CRAIG A BINGMAN 
My guess would be that the chains were prepared under reducing conditions, 
mixed, and then allowed to oxidize. The surface adduct could easily form during 
this process.

On Mar 27, 2020, at 4:38 PM, Cowan, Richard H. (Dr.) 
mailto:rc...@leicester.ac.uk>> wrote:

Hi,

The Fab constructs have a c-terminal cysteines on both the heavy and light 
chains, which should form a disulphide. Adding reducing agent to the 
purification of the protein would potentially reduce this disulphide, possible 
causing issues the stability and heterogeneity? At least that's my 
understanding?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Paul Emsley 
mailto:pems...@mrc-lmb.cam.ac.uk>>
Sent: 27 March 2020 21:33
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Covalent Cysteine Aduct


On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:


  although [BME] seems unlikely, since the crystallized protein is a Fab.


I don't follow.



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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Mark Wilson 
Hi Richard,
Something that hasn’t been mentioned yet that might help settle the issue
is to calculate an anomalous difference map with your I+, I- unmerged
data.  Even if you collect data far from the S edge, it is often possible
to see positive anomalous difference density for S atoms in maps at this
resolution (and with good phases from the refined model).  In the CME case
that Jack Tanner and other mentioned, you’d expect to see two peaks.
Best regards,
Mark

Mark A. Wilson
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 





On 3/27/20, 4:53 PM, "CCP4 bulletin board on behalf of Cowan, Richard H.
(Dr.)"  wrote:

>
>Hi to All,
>
>
>Whilst this discussion has been going on, I've been carrying on with the
>work on this, and have now replaced the offending cysteine with CME. It
>seems to refine pretty well, fitting the density well, and the b-factors
>stay similar to those for nearby side chains,
> so I'm reasonably happy with it. Now to figure out where the BME came
>from.
>
>
>Thanks to all who have offered suggestions and help. Now to check another
>structure with a related Fab with the same surface cysteine, this time
>heavily annisotropic, and in complex with the target, at 2.7A at best!
>
>
>Thanks,
>
>
>
>Dr Richard Cowan
>Research Associate
> 
>HWLSB 1/05
>Department of Biochemistry
>University of Leicester
>Lancaster Road
>Leicester, LE1 9HN, U.K.
> 
>Phone +44 (0) 116 229 7077
>
>
>
>
>
>
>________
>From: Mark J van Raaij 
>Sent: 27 March 2020 21:46
>To: Cowan, Richard H. (Dr.) 
>Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>
>I agree, but in my opinion the density is quite clear for a disulphide,
>so I would bet beta-mercaptoethanol *was* added at some step (perhaps
>even by accident). Even if it's not considered best practice, you seem to
> have "gotten away with it" and the Fab still crystallised ok.
>
>Mark J van Raaij
>Dpto de Estructura de Macromoleculas
>Centro Nacional de Biotecnologia - CSIC
>calle Darwin 3
>E-28049 Madrid, Spain
>Section Editor Acta Crystallographica F
>https://journals.iucr.org/f/
><https://urldefense.proofpoint.com/v2/url?u=https-3A__eur03.safelinks.prot
>ection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fjournals.iucr.org-252Ff-25
>2F-26data-3D02-257C01-257Crc273-2540leicester.ac.uk-257C4d22be0719f0431dc6
>9608d7d298414d-257Caebecd6a31d44b0195ce8274afe853d9-257C0-257C0-257C637209
>423675980222-26sdata-3DQcQSKYwh-252BJ3a86IwEvh6J3UVOplgLRWdfSRvTL8j1sU-253
>D-26reserved-3D0=DwMFAg=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo=
>pWFylCwRYnT2UZGAgJCqKaJCAAfFi00TZEWmZXJvhsA=iYV__iKAZ10TH0IfgFpthVnqpcpX
>ADEZsn1BKx3j4EI=Ca2a6GjDTkPqtBnpDQfjcHriOQXlKDuhn2vLvXO6iMI=>
>
>
>
>
>
>
>
>
>
>
>
>
>On 27 Mar 2020, at 22:38, Cowan, Richard H. (Dr.) 
>wrote:
>
>Hi,
>
>
>The Fab constructs have a c-terminal cysteines on both the heavy and
>light chains, which should form a disulphide. Adding reducing agent to
>the purification of the protein would potentially reduce this disulphide,
>possible causing issues the stability and heterogeneity?
> At least that's my understanding?
>
>
>
>Thanks,
>
>
>
>Dr Richard Cowan
>Research Associate
> 
>HWLSB 1/05
>Department of Biochemistry
>University of Leicester
>Lancaster Road
>Leicester, LE1 9HN, U.K.
> 
>Phone +44 (0) 116 229 7077
>
>
>
>
>
>
>
>
>From: CCP4 bulletin board  on behalf of Paul
>Emsley 
>Sent: 27 March 2020 21:33
>To: CCP4BB@JISCMAIL.AC.UK 
>Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>
>
>
>On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>
>
>
>
>
>
>  although [BME] seems unlikely, since the crystallized protein is a Fab.
>
>
>
>
>
>
>I don't follow.
>
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
><https://urldefense.proofpoint.com/v2/url?u=https-3A__eur03.safelinks.prot
>ection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.jiscmail.ac.uk-252Fcgi
>-2Dbin-252Fwebadmin-253FSUBED1-253DCCP4BB-2526A-253D1-26data-3D02-257C01-2
>57Crc273-2540leicester.ac.uk-257C4d22be0719f0431dc69608d7d298414d-257Caebe
>cd6a31d44b0195ce8274afe853d9-257C0-257C0-257C637209423675980222-26sdata-3D
>Yky4trFxHWJiSDlMu5Ixtyr2MQf6b4g9Xjiuf36ArWc-253D-26reserved-3D0=DwMFAg
>=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo=pWFylCwRYnT2UZGAgJCqKaJCAAf
>Fi00TZEWmZXJvhsA=iYV__iKAZ10TH0Ifg

Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread John Newitt 
There are several ways to prepare “Fabs”, using the term broadly. I would dig 
deeper in this case to be sure a thiol adduct isn’t possible.

John



> On Mar 27, 2020, at 5:38 PM, Cowan, Richard H. (Dr.)  
> wrote:
> 
> 
> Hi,
> 
> The Fab constructs have a c-terminal cysteines on both the heavy and light 
> chains, which should form a disulphide. Adding reducing agent to the 
> purification of the protein would potentially reduce this disulphide, 
> possible causing issues the stability and heterogeneity? At least that's my 
> understanding? 
> 
> Thanks,
> 
> Dr Richard Cowan
> Research Associate
>  
> HWLSB 1/05
> Department of Biochemistry
> University of Leicester
> Lancaster Road
> Leicester, LE1 9HN, U.K.
>  
> Phone +44 (0) 116 229 7077
> 
> From: CCP4 bulletin board  on behalf of Paul Emsley 
> 
> Sent: 27 March 2020 21:33
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>  
> 
> 
>> On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>> 
>> 
>>   although [BME] seems unlikely, since the crystallized protein is a Fab.
>> 
> 
> I don't follow.
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Cowan, Richard H. (Dr.) 
Hi to All,

Whilst this discussion has been going on, I've been carrying on with the work 
on this, and have now replaced the offending cysteine with CME. It seems to 
refine pretty well, fitting the density well, and the b-factors stay similar to 
those for nearby side chains, so I'm reasonably happy with it. Now to figure 
out where the BME came from.

Thanks to all who have offered suggestions and help. Now to check another 
structure with a related Fab with the same surface cysteine, this time heavily 
annisotropic, and in complex with the target, at 2.7A at best!

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: Mark J van Raaij 
Sent: 27 March 2020 21:46
To: Cowan, Richard H. (Dr.) 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct

I agree, but in my opinion the density is quite clear for a disulphide, so I 
would bet beta-mercaptoethanol *was* added at some step (perhaps even by 
accident). Even if it's not considered best practice, you seem to have "gotten 
away with it" and the Fab still crystallised ok.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fjournals.iucr.org%2Ff%2F=02%7C01%7Crc273%40leicester.ac.uk%7C4d22be0719f0431dc69608d7d298414d%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637209423675980222=QcQSKYwh%2BJ3a86IwEvh6J3UVOplgLRWdfSRvTL8j1sU%3D=0>


On 27 Mar 2020, at 22:38, Cowan, Richard H. (Dr.) 
mailto:rc...@leicester.ac.uk>> wrote:

Hi,

The Fab constructs have a c-terminal cysteines on both the heavy and light 
chains, which should form a disulphide. Adding reducing agent to the 
purification of the protein would potentially reduce this disulphide, possible 
causing issues the stability and heterogeneity? At least that's my 
understanding?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Paul Emsley 
mailto:pems...@mrc-lmb.cam.ac.uk>>
Sent: 27 March 2020 21:33
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Covalent Cysteine Aduct


On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:


  although [BME] seems unlikely, since the crystallized protein is a Fab.


I don't follow.



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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Roger Rowlett 
S-hydroxycysteine can cartainly show up under nonreducing storage or
crystallization conditions, as an artifact.

Cheers,

Roger Rowlett

On Fri, Mar 27, 2020, 5:45 PM Chris Fage  wrote:

> Hi Richard,
>
> I recently observed the sulfenic acid derivative of a cysteine residue
> (S-hydroxy-Cys, ligand ID CSO) in one of my high-resolution maps. Could it
> be possibly be this, H-bonded to a nearby water molecule?
>
> Best wishes,
> Chris
>
>
> On Fri, Mar 27, 2020 at 21:33 Paul Emsley 
> wrote:
>
>>
>> On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>>
>>
>>
>>   although [BME] seems unlikely, since the crystallized protein is a Fab.
>>
>>
>> I don't follow.
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Paul Emsley 


I see. Might there be some discrepancy between best practice and what 
actually happened? ?




On 27/03/2020 21:38, Cowan, Richard H. (Dr.) wrote:



The Fab constructs have a c-terminal cysteines on both the heavy and 
light chains, which should form a disulphide. Adding reducing agent to 
the purification of the protein would potentially reduce this 
disulphide, possible causing issues the stability and heterogeneity? 
At least that's my understanding?








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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Chris Fage 
Hi Richard,

I recently observed the sulfenic acid derivative of a cysteine residue
(S-hydroxy-Cys, ligand ID CSO) in one of my high-resolution maps. Could it
be possibly be this, H-bonded to a nearby water molecule?

Best wishes,
Chris


On Fri, Mar 27, 2020 at 21:33 Paul Emsley  wrote:

>
> On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>
>
>
>   although [BME] seems unlikely, since the crystallized protein is a Fab.
>
>
> I don't follow.
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Cowan, Richard H. (Dr.) 
Hi,

The Fab constructs have a c-terminal cysteines on both the heavy and light 
chains, which should form a disulphide. Adding reducing agent to the 
purification of the protein would potentially reduce this disulphide, possible 
causing issues the stability and heterogeneity? At least that's my 
understanding?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: CCP4 bulletin board  on behalf of Paul Emsley 

Sent: 27 March 2020 21:33
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct



On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:


  although [BME] seems unlikely, since the crystallized protein is a Fab.



I don't follow.




To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Paul Emsley 


On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:



although [BME] seems unlikely, since the crystallized protein is a Fab.



I don't follow.





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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Cowan, Richard H. (Dr.) 
Hi,

The gene was synthesized for the expression construct, so a sequence error 
seems unlikely, but I'll check. There should be mass spec data on the purified 
protein, but our collaborators, who are also shut down for the moment, will 
have to get that for me, as well as sequence data. I don't see a guanidino head 
group, which would match arginine (single base error), and the base changes to 
arrive at lysine or methionine seem unlikely to have passed even a cursory QC. 
I will admit that an surface cysteine in a Fab variable domain does seem to be 
unlikely, but I'm not sure how common it may actually be?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: Gulick, Andrew 
Sent: 27 March 2020 21:14
To: Cowan, Richard H. (Dr.) 
Subject: re: [ccp4bb] Covalent Cysteine Aduct


Also, are you sure of protein sequence. Density looked like a long side chain 
(Lys or Arg) so always worth considering.

Best,

Andrew







--

Andrew M. Gulick, 
Ph.D.<https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmedicine.buffalo.edu%2Fcontent%2Fmedicine%2Ffaculty%2Fprofile.html%3Fubit%3Damgulick=02%7C01%7Crc273%40LEICESTER.AC.UK%7C9758cfe572d44a68177b08d7d293db66%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C1%7C637209404794807376=%2F%2FLNQyUrw6ipa3B39U9Mb%2BEsoHRnoqApKMBcarsAtbA%3D=0>

Associate Professor

Department of Structural Biology

Jacobs School of Medicine and Biomedical Sciences

University at Buffalo

955 Main Street, Room 5152

Buffalo, New York 14203-1121

office: 716-829-3696

email: amgul...@buffalo.edu<mailto:amgul...@buffalo.edu>







From: CCP4 bulletin board  on behalf of "Cowan, Richard 
H. (Dr.)" 
Reply-To: "Cowan, Richard H. (Dr.)" 
Date: Friday, March 27, 2020 at 5:06 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct



Hi Robbie et al.,



I'm very sure on the crystallization conditions, but I'll check the 
purification for BME, although it seems unlikely, since the crystallized 
protein is a Fab.



What I would be less sure on is the age of the reagents used. Is anyone aware 
of significant breakdown products which might be more reactive, particularly 
the alcohols?



Thanks,



Dr Richard Cowan
Research Associate



HWLSB 1/05

Department of Biochemistry

University of Leicester

Lancaster Road

Leicester, LE1 9HN, U.K.



Phone +44 (0) 116 229 7077





From: robbie_joos...@hotmail.com 
Sent: 27 March 2020 20:57
To: Cowan, Richard H. (Dr.) 
Cc: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct



Are you absolutely sure about the conditions? The blob next to the sulfur looks 
pretty fat so I would guess BME.



Cheers,

Robbie



On 27 Mar 2020 21:32, "Cowan, Richard H. (Dr.)"  wrote:

Hi All,



During the current enforced shutdown, I've been going back over some older data 
and spotted this in one of my structures:



[cid:d2ff-a10c-4f19-9f47-4db92e7620ef]





It's what appears to be a covalent aduct onto a cysteine on the surface 
(originally modeled as 2 waters!). The condition was optimized from the 
Morpheus Screen condition D1, so MES/Imidazole pH 6.5, PEG 500MME, PEG20k and a 
mix of 1,6-Hexanediol, 1-Butanol, 1,2-Propanediol, 2-Propanol, 1,4-Butanediol 
and 1,3-Propanediol. The data has been cut at 1.7A, with good stats.




Has anyone seen anything like this before? what do you think my best bet for 
modeling this is? and aduct of 1,3-propanediol? It looks too short for 
1-butanol or 1,4-butandiol, and it's linear so 2-propanol and 1,2-propanediol 
seem unlikely.




Thanks,



Dr Richard Cowan
Research Associate



HWLSB 1/05

Department of Biochemistry

University of Leicester

Lancaster Road

Leicester, LE1 9HN, U.K.



Phone +44 (0) 116 229 7077







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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Cowan, Richard H. (Dr.) 
Hi,

The density is very much a linear chain, without side branches. That would lead 
me to discount a DTT adduct, since I can't see anywhere for the hydroxls to go?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: Petri Kursula 
Sent: 27 March 2020 21:13
To: Cowan, Richard H. (Dr.) 
Cc: CCP4BB@jiscmail.ac.uk 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct

Looks like DTT adduct to me; but then again, if you did not have it, it 
probably is not.
Petri

Petri Kursula
--
Professor
--
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula<https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.uib.no%2Fen%2Frg%2Fpetrikursula=02%7C01%7Crc273%40leicester.ac.uk%7C5bd2d6eed27e46e7fa0908d7d293afd2%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637209404081752652=XJzM3H1DJS4TGdmEMzAryNn2YQYpuxcoVkmp9u6sric%3D=0>
petri.kurs...@uib.no
--
Faculty of Biochemistry and Molecular Medicine
Biocenter Oulu
University of Oulu, Finland
--





On 27 Mar 2020, at 22:06, Cowan, Richard H. (Dr.) 
mailto:rc...@leicester.ac.uk>> wrote:

Hi Robbie et al.,

I'm very sure on the crystallization conditions, but I'll check the 
purification for BME, although it seems unlikely, since the crystallized 
protein is a Fab.

What I would be less sure on is the age of the reagents used. Is anyone aware 
of significant breakdown products which might be more reactive, particularly 
the alcohols?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: robbie_joos...@hotmail.com<mailto:robbie_joos...@hotmail.com> 
mailto:robbie_joos...@hotmail.com>>
Sent: 27 March 2020 20:57
To: Cowan, Richard H. (Dr.) 
mailto:rc...@leicester.ac.uk>>
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Covalent Cysteine Aduct

Are you absolutely sure about the conditions? The blob next to the sulfur looks 
pretty fat so I would guess BME.

Cheers,
Robbie

On 27 Mar 2020 21:32, "Cowan, Richard H. (Dr.)" 
mailto:rc...@leicester.ac.uk>> wrote:
Hi All,

During the current enforced shutdown, I've been going back over some older data 
and spotted this in one of my structures:

[cid:d2ff-a10c-4f19-9f47-4db92e7620ef]


It's what appears to be a covalent aduct onto a cysteine on the surface 
(originally modeled as 2 waters!). The condition was optimized from the 
Morpheus Screen condition D1, so MES/Imidazole pH 6.5, PEG 500MME, PEG20k and a 
mix of 1,6-Hexanediol, 1-Butanol, 1,2-Propanediol, 2-Propanol, 1,4-Butanediol 
and 1,3-Propanediol. The data has been cut at 1.7A, with good stats.

Has anyone seen anything like this before? what do you think my best bet for 
modeling this is? and aduct of 1,3-propanediol? It looks too short for 
1-butanol or 1,4-butandiol, and it's linear so 2-propanol and 1,2-propanediol 
seem unlikely.

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Petri Kursula 
Looks like DTT adduct to me; but then again, if you did not have it, it 
probably is not. 
Petri

Petri Kursula
--
Professor 
--
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula
petri.kurs...@uib.no
--
Faculty of Biochemistry and Molecular Medicine
Biocenter Oulu
University of Oulu, Finland
--





> On 27 Mar 2020, at 22:06, Cowan, Richard H. (Dr.)  
> wrote:
> 
> Hi Robbie et al.,
> 
> I'm very sure on the crystallization conditions, but I'll check the 
> purification for BME, although it seems unlikely, since the crystallized 
> protein is a Fab.
> 
> What I would be less sure on is the age of the reagents used. Is anyone aware 
> of significant breakdown products which might be more reactive, particularly 
> the alcohols?
> 
> Thanks,
> 
> Dr Richard Cowan
> Research Associate
>  <> 
> HWLSB 1/05
> Department of Biochemistry
> University of Leicester
> Lancaster Road
> Leicester, LE1 <> 9HN <>, U.K.
>  
> Phone +44 (0) 116 229 7077
> 
> From: robbie_joos...@hotmail.com 
> Sent: 27 March 2020 20:57
> To: Cowan, Richard H. (Dr.) 
> Cc: CCP4BB@JISCMAIL.AC.UK 
> Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>  
> Are you absolutely sure about the conditions? The blob next to the sulfur 
> looks pretty fat so I would guess BME.
> 
> Cheers,
> Robbie
> 
> On 27 Mar 2020 21:32, "Cowan, Richard H. (Dr.)"  wrote:
> Hi All,
> 
> During the current enforced shutdown, I've been going back over some older 
> data and spotted this in one of my structures:
> 
> 
> 
> 
> It's what appears to be a covalent aduct onto a cysteine on the surface 
> (originally modeled as 2 waters!). The condition was optimized from the 
> Morpheus Screen condition D1, so MES/Imidazole pH 6.5, PEG 500MME, PEG20k and 
> a mix of 1,6-Hexanediol, 1-Butanol, 1,2-Propanediol, 2-Propanol, 
> 1,4-Butanediol and 1,3-Propanediol. The data has been cut at 1.7A, with good 
> stats.
> 
> Has anyone seen anything like this before? what do you think my best bet for 
> modeling this is? and aduct of 1,3-propanediol? It looks too short for 
> 1-butanol or 1,4-butandiol, and it's linear so 2-propanol and 1,2-propanediol 
> seem unlikely.
> 
> Thanks,
> 
> Dr Richard Cowan
> Research Associate
>  
> HWLSB 1/05
> Department of Biochemistry
> University of Leicester
> Lancaster Road
> Leicester, LE1 9HN, U.K.
>  
> Phone +44 (0) 116 229 7077
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7Crc273%40LEICESTER.AC.UK%7Ca21cb2bb3ebe45fdcf8e08d7d2917a35%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637209394570788374=i4cJYTIxaA3NqT1u%2FscLnLR3OWxghkRLAiQ2U8zC0kI%3D=0>
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Cowan, Richard H. (Dr.) 
Hi Robbie et al.,

I'm very sure on the crystallization conditions, but I'll check the 
purification for BME, although it seems unlikely, since the crystallized 
protein is a Fab.

What I would be less sure on is the age of the reagents used. Is anyone aware 
of significant breakdown products which might be more reactive, particularly 
the alcohols?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: robbie_joos...@hotmail.com 
Sent: 27 March 2020 20:57
To: Cowan, Richard H. (Dr.) 
Cc: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Covalent Cysteine Aduct

Are you absolutely sure about the conditions? The blob next to the sulfur looks 
pretty fat so I would guess BME.

Cheers,
Robbie

On 27 Mar 2020 21:32, "Cowan, Richard H. (Dr.)"  wrote:
Hi All,

During the current enforced shutdown, I've been going back over some older data 
and spotted this in one of my structures:

[cid:d2ff-a10c-4f19-9f47-4db92e7620ef]


It's what appears to be a covalent aduct onto a cysteine on the surface 
(originally modeled as 2 waters!). The condition was optimized from the 
Morpheus Screen condition D1, so MES/Imidazole pH 6.5, PEG 500MME, PEG20k and a 
mix of 1,6-Hexanediol, 1-Butanol, 1,2-Propanediol, 2-Propanol, 1,4-Butanediol 
and 1,3-Propanediol. The data has been cut at 1.7A, with good stats.

Has anyone seen anything like this before? what do you think my best bet for 
modeling this is? and aduct of 1,3-propanediol? It looks too short for 
1-butanol or 1,4-butandiol, and it's linear so 2-propanol and 1,2-propanediol 
seem unlikely.

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077




To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Robbie P. Joosten 
Are you absolutely sure about the conditions? The blob next to the sulfur looks pretty fat so I would guess BME.Cheers,RobbieOn 27 Mar 2020 21:32, "Cowan, Richard H. (Dr.)"  wrote:

Hi All,




During the current enforced shutdown, I've been going back over some older data and spotted this in one of my structures:













It's what appears to be a covalent aduct onto a cysteine on the surface (originally modeled as 2 waters!). The condition was optimized from the Morpheus
 Screen condition D1, so MES/Imidazole pH 6.5, PEG 500MME, PEG20k and a mix of
 1,6-Hexanediol, 1-Butanol, 1,2-Propanediol, 2-Propanol, 1,4-Butanediol and 1,3-Propanediol. The data has been cut at 1.7A, with good stats.





Has anyone seen anything like this before? what do you think my best bet for modeling
 this is? and aduct of 1,3-propanediol? It looks too short for 1-butanol or 1,4-butandiol, and it's linear so 2-propanol and 1,2-propanediol seem unlikely.





Thanks,








Dr Richard Cowan
Research Associate

 
HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.
 
Phone +44 (0) 116 229 7077









To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Tanner, John J. 
It looks like the density suggests an S-S bond.  Perhaps try CME?

https://www.rcsb.org/ligand/CME


John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
University of Missouri
117 Schweitzer Hall
503 S. College Ave.
Columbia, MO 65211
Phone: 573-884-1280
Fax: 573-882-5635
Email: tanne...@missouri.edu
http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A






On Mar 27, 2020, at 3:32 PM, Cowan, Richard H. (Dr.) 
mailto:rc...@leicester.ac.uk>> wrote:

Hi All,

During the current enforced shutdown, I've been going back over some older data 
and spotted this in one of my structures:




It's what appears to be a covalent aduct onto a cysteine on the surface 
(originally modeled as 2 waters!). The condition was optimized from the 
Morpheus Screen condition D1, so MES/Imidazole pH 6.5, PEG 500MME, PEG20k and a 
mix of 1,6-Hexanediol, 1-Butanol, 1,2-Propanediol, 2-Propanol, 1,4-Butanediol 
and 1,3-Propanediol. The data has been cut at 1.7A, with good stats.

Has anyone seen anything like this before? what do you think my best bet for 
modeling this is? and aduct of 1,3-propanediol? It looks too short for 
1-butanol or 1,4-butandiol, and it's linear so 2-propanol and 1,2-propanediol 
seem unlikely.

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077



To unsubscribe from the CCP4BB list, click the following link:
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