Re: [ccp4bb] CYS modification and choice of PEG
Dear Antonio, we have seen this type of modifications in some of our structures. The modification of the cysteine (to cysteine-sulfenic, sulfinic or sulfonic acid) usually arises from exposure to oxygen during crystallization. We managed to prevent this by either adding TCEP to the protein buffer and/or setting the crystallization trays in an anaerobic chamber. This modification is, to the best of our knowledge, irreversible. Have you tried the cysteine in a (CYS+CSU) alternate conformation (meaning that some molecules got oxidized and some weren't)? Regarding the PEG, you can try PEG, PGE or PG4 which are smaller than PE4. HTH, Jose
Re: [ccp4bb] CYS modification and choice of PEG
If you were collecting at a long-ish wavelength (for NaI phasing, perhaps?) you might be able to see a peak there in an anomalous difference Fourier map. This would be a pretty cool discovery! JPK From: Keller, Jacob Sent: Wednesday, May 17, 2017 4:52 PM To: Antonio Ariza <antonio.ar...@path.ox.ac.uk>; CCP4BB@JISCMAIL.AC.UK Subject: RE: CYS modification and choice of PEG Where would it get the sulphate to make sulphonate? Was there sulphate somewhere in the purification? Maybe it's a phosphate gotten off the FMN? I guess the phospho-cys bond might be a bit longer? JPK Analyst.<https://www.ncbi.nlm.nih.gov/pubmed/25011562> 2014 Sep 7;139(17):4118-23. doi: 10.1039/c4an00724g. Puzzling over protein cysteine phosphorylation--assessment of proteomic tools for S-phosphorylation profiling. Buchowiecka AK<https://www.ncbi.nlm.nih.gov/pubmed/?term=Buchowiecka%20AK%5BAuthor%5D=true_uid=25011562>1. Author information<https://www.ncbi.nlm.nih.gov/pubmed/25011562> Abstract Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Antonio Ariza Sent: Wednesday, May 17, 2017 4:04 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] CYS modification and choice of PEG I haven't asked anything for a loong time, so here are a couple of question "for the honourable members of the esteemed CCP4 bulletin board" ... or as I'd usually say: "for y'all". ;) 1) I have this modified CYS in one of the structures I'm working on and ... I'm quite unhappy with it (see attached pics). At first I thought it was cacodylate ... but alas, no cacodylate was used during purification or crystallisation. So I looked up possible modifications on CYS residues and I came up with cysteine-s-sulfonic acid (CSU). This looks good in principle but it doesn't quite fit the electron density as the bond between the two sulphur atoms is too short. The average length for an S-S bond is about 2.05 Ang and refmac refines this one to 1.97 Ang, but it looks like it should be at least 2.5 Ang to sit correctly in the electron density. Also, there is some negative density in there, suggesting that maybe it's something with fewer electrons than a sulfonic acid ... or maybe it has less than 100% occupancy. Any suggestions? The condition contains TRIS, bicine, NaCl, NaFl, NaI, DTT, FMN, PEG 500 MME and PEG 20,000. 2) There are two partial PEG molecules in this structure. I've initially modeled two PEG 400 (PE4) molecules into the density (simply because I remembered the 3 -letter code for it) and removed the excess atoms from them. However, since there is a mixture of PEG 500 MME and PEG 20,000 in the condition, what would you recommend I use instead of PE4? Cheers, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5>
Re: [ccp4bb] CYS modification and choice of PEG
Where would it get the sulphate to make sulphonate? Was there sulphate somewhere in the purification? Maybe it's a phosphate gotten off the FMN? I guess the phospho-cys bond might be a bit longer? JPK Analyst.<https://www.ncbi.nlm.nih.gov/pubmed/25011562> 2014 Sep 7;139(17):4118-23. doi: 10.1039/c4an00724g. Puzzling over protein cysteine phosphorylation--assessment of proteomic tools for S-phosphorylation profiling. Buchowiecka AK<https://www.ncbi.nlm.nih.gov/pubmed/?term=Buchowiecka%20AK%5BAuthor%5D=true_uid=25011562>1. Author information<https://www.ncbi.nlm.nih.gov/pubmed/25011562> Abstract Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Antonio Ariza Sent: Wednesday, May 17, 2017 4:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] CYS modification and choice of PEG I haven't asked anything for a loong time, so here are a couple of question "for the honourable members of the esteemed CCP4 bulletin board" ... or as I'd usually say: "for y'all". ;) 1) I have this modified CYS in one of the structures I'm working on and ... I'm quite unhappy with it (see attached pics). At first I thought it was cacodylate ... but alas, no cacodylate was used during purification or crystallisation. So I looked up possible modifications on CYS residues and I came up with cysteine-s-sulfonic acid (CSU). This looks good in principle but it doesn't quite fit the electron density as the bond between the two sulphur atoms is too short. The average length for an S-S bond is about 2.05 Ang and refmac refines this one to 1.97 Ang, but it looks like it should be at least 2.5 Ang to sit correctly in the electron density. Also, there is some negative density in there, suggesting that maybe it's something with fewer electrons than a sulfonic acid ... or maybe it has less than 100% occupancy. Any suggestions? The condition contains TRIS, bicine, NaCl, NaFl, NaI, DTT, FMN, PEG 500 MME and PEG 20,000. 2) There are two partial PEG molecules in this structure. I've initially modeled two PEG 400 (PE4) molecules into the density (simply because I remembered the 3 -letter code for it) and removed the excess atoms from them. However, since there is a mixture of PEG 500 MME and PEG 20,000 in the condition, what would you recommend I use instead of PE4? Cheers, Tony -- Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5>
Re: [ccp4bb] CYS modification and choice of PEG
On Wednesday, 17 May, 2017 20:03:42 Antonio Ariza wrote: > I haven't asked anything for a loong time, so here are a couple of > question "for the honourable members of the esteemed CCP4 bulletin board" ... > or as I'd usually say: "for y'all". ;) > > 1) I have this modified CYS in one of the structures I'm working on and ... > I'm quite unhappy with it (see attached pics). At first I thought it was > cacodylate ... but alas, no cacodylate was used during purification or > crystallisation. So I looked up possible modifications on CYS residues and I > came up with cysteine-s-sulfonic acid (CSU). This looks good in principle but > it doesn't quite fit the electron density as the bond between the two sulphur > atoms is too short. The average length for an S-S bond is about 2.05 Ang and > refmac refines this one to 1.97 Ang, but it looks like it should be at least > 2.5 Ang to sit correctly in the electron density. Also, there is some > negative density in there, suggesting that maybe it's something with fewer > electrons than a sulfonic acid ... or maybe it has less than 100% occupancy. > Any suggestions? Could it be that a cloning error introduced a Cys->Arg mutation? -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg MS 357742, University of Washington, Seattle 98195-7742
