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 <amgul...@buffalo.edu> Sent: 27 March 2020 21:14 To: Cowan, Richard H. (Dr.) <rc...@leicester.ac.uk> 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&data=02%7C01%7Crc273%40LEICESTER.AC.UK%7C9758cfe572d44a68177b08d7d293db66%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C1%7C637209404794807376&sdata=%2F%2FLNQyUrw6ipa3B39U9Mb%2BEsoHRnoqApKMBcarsAtbA%3D&reserved=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 <CCP4BB@JISCMAIL.AC.UK> on behalf of "Cowan, Richard H. (Dr.)" <rc...@leicester.ac.uk> Reply-To: "Cowan, Richard H. (Dr.)" <rc...@leicester.ac.uk> Date: Friday, March 27, 2020 at 5:06 PM To: "CCP4BB@JISCMAIL.AC.UK" <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 <robbie_joos...@hotmail.com> Sent: 27 March 2020 20:57 To: Cowan, Richard H. (Dr.) <rc...@leicester.ac.uk> Cc: CCP4BB@JISCMAIL.AC.UK <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.)" <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:ddddd2ff-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: 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%7C9758cfe572d44a68177b08d7d293db66%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C1%7C637209404794807376&sdata=CMVlivfCHdlT3CQJo2xIXtE1rOO4rpESY0BJ2gZyoe0%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%7C9758cfe572d44a68177b08d7d293db66%7Caebecd6a31d44b0195ce8274afe853d9%7C0%7C1%7C637209404794817370&sdata=zIpefQU%2Fe6OMf0nyKEu%2BtbfD8YhzUPWfCydbZOyp2bA%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
