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
[log in to unmask]
On 3/27/20, 4:53 PM, "CCP4 bulletin board on behalf of Cowan, Richard H.
(Dr.)" <[log in to unmask] on behalf of [log in to unmask]> 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 <[log in to unmask]>
>Sent: 27 March 2020 21:46
>To: Cowan, Richard H. (Dr.) <[log in to unmask]>
>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&d=DwMFAg&c=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo&r=
>pWFylCwRYnT2UZGAgJCqKaJCAAfFi00TZEWmZXJvhsA&m=iYV__iKAZ10TH0IfgFpthVnqpcpX
>ADEZsn1BKx3j4EI&s=Ca2a6GjDTkPqtBnpDQfjcHriOQXlKDuhn2vLvXO6iMI&e=>
>
>
>
>
>
>
>
>
>
>
>
>
>On 27 Mar 2020, at 22:38, Cowan, Richard H. (Dr.) <[log in to unmask]>
>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 <[log in to unmask]> on behalf of Paul
>Emsley <[log in to unmask]>
>Sent: 27 March 2020 21:33
>To: [log in to unmask] <[log in to unmask]>
>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&A=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&d=DwMFAg&c
>=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo&r=pWFylCwRYnT2UZGAgJCqKaJCAAf
>Fi00TZEWmZXJvhsA&m=iYV__iKAZ10TH0IfgFpthVnqpcpXADEZsn1BKx3j4EI&s=SJBFc6JbO
>Z8aPDgQ4wyx7LZh-C_y9xEnS2okHG24CFE&e=>
>
>
>________________________________________
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=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
>cd6a31d44b0195ce8274afe853d9-257C0-257C0-257C637209423675990177-26sdata-3D
>UNAjd-252FEyknJHkIArWRjMcUkihlIzq0P7nCAjqTw-252FLIk-253D-26reserved-3D0&d=
>DwMFAg&c=Cu5g146wZdoqVuKpTNsYHeFX_rg6kWhlkLF8Eft-wwo&r=pWFylCwRYnT2UZGAgJC
>qKaJCAAfFi00TZEWmZXJvhsA&m=iYV__iKAZ10TH0IfgFpthVnqpcpXADEZsn1BKx3j4EI&s=a
>z8bfKI7pOSYd9s6aza5gY6OG5vsQ9IL55Mu1HLXx2o&e=>
>
>
>
>
>
>
>
>
>________________________________________
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
><https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_c
>gi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFAg&c=Cu5g146wZdoqVuKpTNs
>YHeFX_rg6kWhlkLF8Eft-wwo&r=pWFylCwRYnT2UZGAgJCqKaJCAAfFi00TZEWmZXJvhsA&m=i
>YV__iKAZ10TH0IfgFpthVnqpcpXADEZsn1BKx3j4EI&s=LJ55IT2TP96v-XrQvv7npa0ROZz7a
>5wbkd4LDJjqNKM&e=>
########################################################################
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
|