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 <mjvanra...@cnb.csic.es> Sent: 27 March 2020 21:46 To: Cowan, Richard H. (Dr.) <rc...@leicester.ac.uk> 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&data=02%7C01%7Crc273%40leicester.ac.uk%7C4d22be0719f0431dc69608d7d298414d%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637209423675980222&sdata=QcQSKYwh%2BJ3a86IwEvh6J3UVOplgLRWdfSRvTL8j1sU%3D&reserved=0> On 27 Mar 2020, at 22:38, Cowan, Richard H. (Dr.) <rc...@leicester.ac.uk<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 <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Paul Emsley <pems...@mrc-lmb.cam.ac.uk<mailto:pems...@mrc-lmb.cam.ac.uk>> Sent: 27 March 2020 21:33 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <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. ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7Crc273%40leicester.ac.uk%7C4d22be0719f0431dc69608d7d298414d%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637209423675980222&sdata=Yky4trFxHWJiSDlMu5Ixtyr2MQf6b4g9Xjiuf36ArWc%3D&reserved=0> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7Crc273%40leicester.ac.uk%7C4d22be0719f0431dc69608d7d298414d%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C0%7C637209423675990177&sdata=UNAjd%2FEyknJHkIArWRjMcUkihlIzq0P7nCAjqTw%2FLIk%3D&reserved=0> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
