Re: [ccp4bb] CSX
Dear All; Thank you very much for your comments and advices. My protein is homo-tetramer. Based on difference density, only one cysteine (among the four catalytic cysteins) has observed two positive density along SG-group. This one could be modeled into OCS, while the rest will be modeled as non-oxidized. I very much appreciate for all your inputs regards Uma On Fri, May 4, 2012 at 6:09 PM, Savvas Savvides savvas.savvi...@ugent.bewrote: Dear Uma Your modeling of a Cysteine sulfenic group (SOH) is reasonable given the observed difference density but do keep in mind that sulfenic groups are very susceptible to further oxidation to sulfinic and sulfonic acids. Stabilization of sulfenic and sulfinic groups in enzyme active sites is possible as shown crystallographically by Becker et al Nat Struct Biol 5:267-271 (1998). Also make sure that you flip the Histidine in the active site that faces the SOH group to account for a possible hydrogen bond. Best regards Savvas On 04 May 2012, at 20:24, Uma Ratu rosiso2...@gmail.com wrote: Dear All: Thank you very much for your advices and comments. Following your instructions, I am able to change the CYS to CSX. Both Get Monomer and Replace Residue work well. I have the refined conformation CSX (by real space refinment ) attached (named as M-CSX-1). The Fo-Fc map is shown with sigma @2.0. Is this conformation reasonable? Why there are two bond conformation there? Thank you for comments. Uma On Fri, May 4, 2012 at 12:10 PM, Hugo Correia h.corr...@campus.fct.unl.pt wrote: Dear Uma, You can do this using coot. Go to Extensions Modelling Replace Residue... and enter the three letter code. Cheers Hugo Correia 2012/5/4 Uma Ratu rosiso2...@gmail.com Dear All: My protein has a key cysteine residue involved in catalytic activity. The template structure used for the modeling has the same key cysteine. In the template structure, this key cysteine residue is assigned as CSX based on the observation from its electronic density. I compared the electron density from the template as well as my model. I can't tell if the cysteine in my model is oxidized or not. The ones from the template also looks different from each other, although both assigned as CSX. I have the snapshots of these cysteines attached. The ones from my model named as M-, and the ones from the template named as T-. Plus, how to change the residue label from Cys to CSX if the cystein is oxidized? In coot, I could not find such function. Thank you very much for your advice Uma M-CSX-1.tif
Re: [ccp4bb] CSX
Hi Uma, I wish you would have shown negative densities on Fo-Fc maps as well. You protein (and the template one) may end up being a classical case to demonstrate how radiation-induced changes may lead to undesirable interpretations. Especially because it is a Cys and because it is in the active site. Cheers, Nukri Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Uma Ratu Sent: Friday, May 04, 2012 10:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] CSX Dear All: My protein has a key cysteine residue involved in catalytic activity. The template structure used for the modeling has the same key cysteine. In the template structure, this key cysteine residue is assigned as CSX based on the observation from its electronic density. I compared the electron density from the template as well as my model. I can't tell if the cysteine in my model is oxidized or not. The ones from the template also looks different from each other, although both assigned as CSX. I have the snapshots of these cysteines attached. The ones from my model named as M-, and the ones from the template named as T-. Plus, how to change the residue label from Cys to CSX if the cystein is oxidized? In coot, I could not find such function. Thank you very much for your advice Uma
Re: [ccp4bb] CSX
Uma, It is possible you have either S-hydroxycysteine (CSO) or S-oxycysteine (CSX). The only difference, crystallographically, is that the CSO S-O bond is about 1.78A and the CSX S-O bond is about 1.50A. You may not be able to differentiate this difference in your electron density maps. A SH - HOH contact should be considerably longer than 1.8 A between heavy atoms, certainly more like 3.0A+/-. You can easily substitute either CSO or CSX for CYS in Coot by deleting the CYS residue and then using File...Get Monomer and typing in either CSO or CSX. (Both CSO and CSX are in the Refmac monomer library.) Rotate things into place and do a real-space refinement to get things closer, and you should be all set for the next Refmac round of refinement. There may be a more elegant way of doing this in Coot, but this will work. If you want to crudely see if your refinement with CSO or CSX is reasonable, create an omit map by setting the occupancy of the oxygen atom to zero. You should see some nicely centered positive difference density on the oxygen atom. I encountered a surprise CSO residue when solving the structure of 3UAO. The protein was stored by a collaborator in a non-reducing medium, resulting in the oxidation of the active site Cys residue. (The only Cys in the protein that oxidized, natch.) The electron density near Cys was too close to be a water molecule, but CSO modeled nicely. The electron-density-suggested bond distance was about 1.70 A, so CSO looked better than CSX, but I bet both would model OK. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/4/2012 11:00 AM, Uma Ratu wrote: Dear All: My protein has a key cysteine residue involved in catalytic activity. The template structure used for the modeling has the same key cysteine. In the template structure, this key cysteine residue is assigned as CSX based on the observation from its electronic density. I compared the electron density from the template as well as my model. I can't tell if the cysteine in my model is oxidized or not. The ones from the template also looks different from each other, although both assigned as CSX. I have the snapshots of these cysteines attached. The ones from my model named as M-, and the ones from the template named as T-. Plus, how to change the residue label from Cys to CSX if the cystein is oxidized? In coot, I could not find such function. Thank you very much for your advice Uma
Re: [ccp4bb] CSX
Dear Uma Your modeling of a Cysteine sulfenic group (SOH) is reasonable given the observed difference density but do keep in mind that sulfenic groups are very susceptible to further oxidation to sulfinic and sulfonic acids. Stabilization of sulfenic and sulfinic groups in enzyme active sites is possible as shown crystallographically by Becker et al Nat Struct Biol 5:267-271 (1998). Also make sure that you flip the Histidine in the active site that faces the SOH group to account for a possible hydrogen bond. Best regards Savvas On 04 May 2012, at 20:24, Uma Ratu rosiso2...@gmail.com wrote: Dear All: Thank you very much for your advices and comments. Following your instructions, I am able to change the CYS to CSX. Both Get Monomer and Replace Residue work well. I have the refined conformation CSX (by real space refinment ) attached (named as M-CSX-1). The Fo-Fc map is shown with sigma @2.0. Is this conformation reasonable? Why there are two bond conformation there? Thank you for comments. Uma On Fri, May 4, 2012 at 12:10 PM, Hugo Correia h.corr...@campus.fct.unl.pt wrote: Dear Uma, You can do this using coot. Go to Extensions Modelling Replace Residue... and enter the three letter code. Cheers Hugo Correia 2012/5/4 Uma Ratu rosiso2...@gmail.com Dear All: My protein has a key cysteine residue involved in catalytic activity. The template structure used for the modeling has the same key cysteine. In the template structure, this key cysteine residue is assigned as CSX based on the observation from its electronic density. I compared the electron density from the template as well as my model. I can't tell if the cysteine in my model is oxidized or not. The ones from the template also looks different from each other, although both assigned as CSX. I have the snapshots of these cysteines attached. The ones from my model named as M-, and the ones from the template named as T-. Plus, how to change the residue label from Cys to CSX if the cystein is oxidized? In coot, I could not find such function. Thank you very much for your advice Uma M-CSX-1.tif
