Re: [ccp4bb] Fo-Fc density close to cysteine residue
I think this is very wise. It can be better to say that you don't know then to guess if people will take your answer for granted. Cheers, Robbie On 11 Jul 2019 11:09, Raz Zarivach <33371661b7b1-dmarc-requ...@jiscmail.ac.uk> wrote: Hi All With so many alternative suggestions, wouldn't be better to leave it without guessing the wrong molecule? Raz On 11/07/2019 10:55, Anthony Addlagatta wrote: > For some reason if your sulfur on cysteine becomes sulfide anion, you can > model sodium/potassium. Distance seems to be optimum for S...Na contact. > Since you do not see any anomalous signal, this is one possibility. I also > see small blobs around which may resolve to be water molecules. > > Anthony > > - Original Message - > From: "hideaki niwa" > To: CCP4BB@JISCMAIL.AC.UK > Sent: Thursday, July 11, 2019 12:22:39 PM > Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue > > Hi Sergei, > > At a first glance I thought so too, but if it is an arsenic atom there > should be a strong anomalous signal. Also the distance 2.5A seems a bit > long. > > Hideaki Niwa > > RIKEN Center for Biosystems Dynamics Research > Yokohama, 230-0045 JAPAN > > On 2019/07/10 23:45, Sergei Strelkov wrote: >> Dear Lumbini, >> >> >> Certainly not a proof but your density looks like >> (di)methyl-arsinoyl-cysteine (PDB residue type CAF). This modification >> can happen when the protein has been exposed to cacodylate buffer. You >> can find such residues (and maps) in the PDB entries 3LPT or 5MDI. >> >> >> Best wishes, >> >> Sergei >> >> >> Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of >> Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat >> 49 bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 >> 29 41 32 Lab pages: http://pharm.kuleuven.be/Biocrystallography >> >> -------------------- >> *From:* CCP4 bulletin board on behalf of Lumbini >> Yadav >> *Sent:* Wednesday, July 10, 2019 13:12 >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* Re: [ccp4bb] Fo-Fc density close to cysteine residue >> Thank you for the valuable input >> >> On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson >> mailto:eleanor.dod...@york.ac.uk>> wrote: >> >> Well - that more or less proves the residue is a CYS - there is a >> peak in the PHAN map right on the S. >> And that the extra density does not contain an anomalous scatterer >> so is probably not a metal or a sulphate or... >> >> But that still doesnt explain WHAT it is . Sorry not to be more help.. >> >> Eleanor >> >> On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav > <mailto:lumbin...@gmail.com>> wrote: >> >> No I am using ccp4i. I tried doing SAD refinement in refmac and >> the output image is attached below . >> I do not seen density near cysteine that was visible in Fo-Fc map >> >> On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson >> mailto:eleanor.dod...@york.ac.uk>> >> wrote: >> >> The key word for refmac is ANOM MAPONLY >> Are you using GUI2? >> Eleanor >> >> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav >> mailto:lumbin...@gmail.com>> wrote: >> >> I have soaked my crystals in?? sodium dithionite a >> reducing agent. I have not done mass spec but sequence >> is confirmed >> >> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel >> > <mailto:dbon...@som.umaryland.edu>> wrote: >> >> Have you mass-speced the protein before >> crystallization to make sure it wasn???t derivatized >> during expression and/or purification, or compared >> the mass spec of the crystals verses purified >> protein? Any fancy reagents or other reductants used >> during purification? >> >> >> >> What about S-Acetyl-cysteine (3-letter code: SCY). >> >> __ __ >> >> Best, >> >> __ __ >> >> Dan >> >> __ __ >> >> Daniel
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Hi All With so many alternative suggestions, wouldn't be better to leave it without guessing the wrong molecule? Raz On 11/07/2019 10:55, Anthony Addlagatta wrote: For some reason if your sulfur on cysteine becomes sulfide anion, you can model sodium/potassium. Distance seems to be optimum for S...Na contact. Since you do not see any anomalous signal, this is one possibility. I also see small blobs around which may resolve to be water molecules. Anthony - Original Message - From: "hideaki niwa" To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 11, 2019 12:22:39 PM Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue Hi Sergei, At a first glance I thought so too, but if it is an arsenic atom there should be a strong anomalous signal. Also the distance 2.5A seems a bit long. Hideaki Niwa RIKEN Center for Biosystems Dynamics Research Yokohama, 230-0045 JAPAN On 2019/07/10 23:45, Sergei Strelkov wrote: Dear Lumbini, Certainly not a proof but your density looks like (di)methyl-arsinoyl-cysteine (PDB residue type CAF). This modification can happen when the protein has been exposed to cacodylate buffer. You can find such residues (and maps) in the PDB entries 3LPT or 5MDI. Best wishes, Sergei Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab pages: http://pharm.kuleuven.be/Biocrystallography *From:* CCP4 bulletin board on behalf of Lumbini Yadav *Sent:* Wednesday, July 10, 2019 13:12 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Fo-Fc density close to cysteine residue Thank you for the valuable input On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S. And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or... But that still doesnt explain WHAT it is . Sorry not to be more help.. Eleanor On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below . I do not seen density near cysteine that was visible in Fo-Fc map On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: The key word for refmac is ANOM MAPONLY Are you using GUI2? Eleanor On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: I have soaked my crystals in?? sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel mailto:dbon...@som.umaryland.edu>> wrote: Have you mass-speced the protein before crystallization to make sure it wasn???t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine (3-letter code: SCY). __ __ Best, __ __ Dan __ __ Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 __ __ __ __ *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>] *On Behalf Of *Lumbini Yadav *Sent:* Tuesday, July 09, 2019 5:22 AM *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue __ __ Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) i
Re: [ccp4bb] Fo-Fc density close to cysteine residue
For some reason if your sulfur on cysteine becomes sulfide anion, you can model sodium/potassium. Distance seems to be optimum for S...Na contact. Since you do not see any anomalous signal, this is one possibility. I also see small blobs around which may resolve to be water molecules. Anthony - Original Message - From: "hideaki niwa" To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 11, 2019 12:22:39 PM Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue Hi Sergei, At a first glance I thought so too, but if it is an arsenic atom there should be a strong anomalous signal. Also the distance 2.5A seems a bit long. Hideaki Niwa RIKEN Center for Biosystems Dynamics Research Yokohama, 230-0045 JAPAN On 2019/07/10 23:45, Sergei Strelkov wrote: > Dear Lumbini, > > > Certainly not a proof but your density looks like > (di)methyl-arsinoyl-cysteine (PDB residue type CAF). This modification > can happen when the protein has been exposed to cacodylate buffer. You > can find such residues (and maps) in the PDB entries 3LPT or 5MDI. > > > Best wishes, > > Sergei > > > Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of > Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat > 49 bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 > 29 41 32 Lab pages: http://pharm.kuleuven.be/Biocrystallography > > > *From:* CCP4 bulletin board on behalf of Lumbini > Yadav > *Sent:* Wednesday, July 10, 2019 13:12 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Fo-Fc density close to cysteine residue > Thank you for the valuable input > > On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson > mailto:eleanor.dod...@york.ac.uk>> wrote: > > Well - that more or less proves the residue is a CYS - there is a > peak in the PHAN map right on the S. > And that the extra density does not contain an anomalous scatterer > so is probably not a metal or a sulphate or... > > But that still doesnt explain WHAT it is . Sorry not to be more help.. > > Eleanor > > On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav <mailto:lumbin...@gmail.com>> wrote: > > No I am using ccp4i. I tried doing SAD refinement in refmac and > the output image is attached below . > I do not seen density near cysteine that was visible in Fo-Fc map > > On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson > mailto:eleanor.dod...@york.ac.uk>> > wrote: > > The key word for refmac is ANOM MAPONLY > Are you using GUI2? > Eleanor > > On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav > mailto:lumbin...@gmail.com>> wrote: > > I have soaked my crystals in sodium dithionite a > reducing agent. I have not done mass spec but sequence > is confirmed > > On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel > <mailto:dbon...@som.umaryland.edu>> wrote: > > Have you mass-speced the protein before > crystallization to make sure it wasn’t derivatized > during expression and/or purification, or compared > the mass spec of the crystals verses purified > protein? Any fancy reagents or other reductants used > during purification? > > > > What about S-Acetyl-cysteine (3-letter code: SCY). > > __ __ > > Best, > > __ __ > > Dan > > __ __ > > Daniel A Bonsor PhD. > > Sundberg Lab > > Institute of Human Virology > > University of Maryland, Baltimore > > 725 W Lombard Street N370 > > Baltimore > > Maryland > > MD 21201 > > Tel: (410) 706-7457 > > __ __ > > __ __ > > *From:*CCP4 bulletin board > [mailto:CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK>] *On Behalf Of > *Lumbini Yadav > *Sent:* Tuesday, July 09, 2019 5:22 AM > *To:* CCP4BB@JISCMAIL.AC.UK
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Hi Sergei, At a first glance I thought so too, but if it is an arsenic atom there should be a strong anomalous signal. Also the distance 2.5A seems a bit long. Hideaki Niwa RIKEN Center for Biosystems Dynamics Research Yokohama, 230-0045 JAPAN On 2019/07/10 23:45, Sergei Strelkov wrote: Dear Lumbini, Certainly not a proof but your density looks like (di)methyl-arsinoyl-cysteine (PDB residue type CAF). This modification can happen when the protein has been exposed to cacodylate buffer. You can find such residues (and maps) in the PDB entries 3LPT or 5MDI. Best wishes, Sergei Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab pages: http://pharm.kuleuven.be/Biocrystallography *From:* CCP4 bulletin board on behalf of Lumbini Yadav *Sent:* Wednesday, July 10, 2019 13:12 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Fo-Fc density close to cysteine residue Thank you for the valuable input On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S. And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or... But that still doesnt explain WHAT it is . Sorry not to be more help.. Eleanor On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below . I do not seen density near cysteine that was visible in Fo-Fc map On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: The key word for refmac is ANOM MAPONLY Are you using GUI2? Eleanor On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: I have soaked my crystals in sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel mailto:dbon...@som.umaryland.edu>> wrote: Have you mass-speced the protein before crystallization to make sure it wasn’t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine (3-letter code: SCY). __ __ Best, __ __ Dan __ __ Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 __ __ __ __ *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>] *On Behalf Of *Lumbini Yadav *Sent:* Tuesday, July 09, 2019 5:22 AM *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue __ __ Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC,CSD, CXM,SCH,CSU) from the library and also SO_3 , SO_2 and peroxide. But in all the screenings we do see some part of Fo-Fc
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Hi Lumbini, There are couple of queries I have before commenting on this issue. 1) how many Cysteine residues are there in your protein 2) have you calculated their solvent accessible surface area ? 3) What is the final concentration of Sodium dithionite ? Cysteine is highly prone to get thiol covalently modified if it is quite accessible to the solvent in its free form. As you know, sodium dithionite is having almost same chemical structure like BME. And you will find N numbers of papers about how BME covalently modifies the accessible cysteine in the protein. It seems to me that it modified by sodium dithionite by air oxidation. Please check the facts and try to fit with manually built derivative of the sodium dithionite with this cysteine residue. Good luck P.P On Tue, 9 Jul 2019, 04:32 Lumbini Yadav, wrote: > Dear all, > > > > We have found a huge Fo-Fc density close to cysteine residue (see attached > image) in the structure with resolution of 1.2A. In the crystallization > condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and > protein was in Tris and NaCl. Before freezing the crystals were soaked in > mother liquor containing sodium dithionite. > > > > I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, > CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in > all the screenings we do see some part of Fo-Fc density unaddressed at 3 > sigma. > > > > Does anyone have an idea about what this density could be? Covalent > modification? > > > > Thanks. > > > > Kind regards, > > Lumbini > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Hi Jakob, If there is radiation damage, for every "new" positive density there should be a negative density in the original position. From the pictures we've seen, it seems unlikely. Best, Nukri On Wed, Jul 10, 2019 at 10:21 AM Keller, Jacob wrote: > How about radiation-damaged/smashed Sulphur? You could test this by > refining occupancy of the cys S. > > > > JPK > > > > + > > Jacob Pearson Keller > > Research Scientist / Looger Lab > > HHMI Janelia Research Campus > > 19700 Helix Dr, Ashburn, VA 20147 > > Desk: (571)209-4000 x3159 > > Cell: (301)592-7004 > > + > > > > The content of this email is confidential and intended for the recipient > specified in message only. It is strictly forbidden to share any part of > this message with any third party, without a written consent of the sender. > If you received this message by mistake, please reply to this message and > follow with its deletion, so that we can ensure such a mistake does not > occur in the future. > > > > *From:* CCP4 bulletin board * On Behalf Of *Lumbini > Yadav > *Sent:* Wednesday, July 10, 2019 7:12 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Fo-Fc density close to cysteine residue > > > > Thank you for the valuable input > > > > On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson > wrote: > > Well - that more or less proves the residue is a CYS - there is a peak in > the PHAN map right on the S. > > And that the extra density does not contain an anomalous scatterer so is > probably not a metal or a sulphate or... > > > > But that still doesnt explain WHAT it is . Sorry not to be more help.. > > > > Eleanor > > > > On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav wrote: > > No I am using ccp4i. I tried doing SAD refinement in refmac and the output > image is attached below . > > I do not seen density near cysteine that was visible in Fo-Fc map > > > > On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson > wrote: > > The key word for refmac is ANOM MAPONLY > > Are you using GUI2? > > Eleanor > > > > On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav wrote: > > I have soaked my crystals in sodium dithionite a reducing agent. I have > not done mass spec but sequence is confirmed > > > > On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel > wrote: > > Have you mass-speced the protein before crystallization to make sure it > wasn’t derivatized during expression and/or purification, or compared the > mass spec of the crystals verses purified protein? Any fancy reagents or > other reductants used during purification? > > What about S-Acetyl-cysteine (3-letter code: SCY). > > > > Best, > > > > Dan > > > > Daniel A Bonsor PhD. > > Sundberg Lab > > Institute of Human Virology > > University of Maryland, Baltimore > > 725 W Lombard Street N370 > > Baltimore > > Maryland > > MD 21201 > > Tel: (410) 706-7457 > > > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Lumbini > Yadav > *Sent:* Tuesday, July 09, 2019 5:22 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue > > > > Dear all, > > > > We have found a huge Fo-Fc density close to cysteine residue (see attached > image) in the structure with resolution of 1.2A. In the crystallization > condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and > protein was in Tris and NaCl. Before freezing the crystals were soaked in > mother liquor containing sodium dithionite. > > > > I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, > CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in > all the screenings we do see some part of Fo-Fc density unaddressed at 3 > sigma. > > > > Does anyone have an idea about what this density could be? Covalent > modification? > > > > Thanks. > > > > > > Kind regards, > > Lumbini > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=Z963G6bopP8xJVXD1Wuy672eSOZyBi1ySdwyyfyUY28=eacDldZlgDNyZ5rJs_4-dTAl4HGX6sPpzGppOB85YNk=> > > > -- > &g
Re: [ccp4bb] Fo-Fc density close to cysteine residue
How about radiation-damaged/smashed Sulphur? You could test this by refining occupancy of the cys S. JPK + Jacob Pearson Keller Research Scientist / Looger Lab HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 Desk: (571)209-4000 x3159 Cell: (301)592-7004 + The content of this email is confidential and intended for the recipient specified in message only. It is strictly forbidden to share any part of this message with any third party, without a written consent of the sender. If you received this message by mistake, please reply to this message and follow with its deletion, so that we can ensure such a mistake does not occur in the future. From: CCP4 bulletin board On Behalf Of Lumbini Yadav Sent: Wednesday, July 10, 2019 7:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue Thank you for the valuable input On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S. And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or... But that still doesnt explain WHAT it is . Sorry not to be more help.. Eleanor On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below . I do not seen density near cysteine that was visible in Fo-Fc map On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: The key word for refmac is ANOM MAPONLY Are you using GUI2? Eleanor On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: I have soaked my crystals in sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel mailto:dbon...@som.umaryland.edu>> wrote: Have you mass-speced the protein before crystallization to make sure it wasn’t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine (3-letter code: SCY). Best, Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Lumbini Yadav Sent: Tuesday, July 09, 2019 5:22 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Fo-Fc density close to cysteine residue Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma. Does anyone have an idea about what this density could be? Covalent modification? Thanks. Kind regards, Lumbini To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=Z963G6bopP8xJVXD1Wuy672eSOZyBi1ySdwyyfyUY28=eacDldZlgDNyZ5rJs_4-dTAl4HGX6sPpzGppOB85YNk=> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=Z963G6bopP8xJVXD1Wuy672eSOZyBi1ySdwyyfyUY28=eacDldZlgDNyZ5rJs_4-dTAl4HGX6sPpzGppOB85YNk=> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=LU6cRtx0xgB8s29tIz9Olw=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI=Z963G6bopP8xJVXD1Wuy672eSOZyBi1ySdwyyfyUY28=eacDldZlgDNyZ5rJs_4-dTAl4HGX6sPpzGppOB85YNk=> T
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Dear Lumbini, Certainly not a proof but your density looks like (di)methyl-arsinoyl-cysteine (PDB residue type CAF). This modification can happen when the protein has been exposed to cacodylate buffer. You can find such residues (and maps) in the PDB entries 3LPT or 5MDI. Best wishes, Sergei Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab pages: http://pharm.kuleuven.be/Biocrystallography<http://pharm.kuleuven.be/anafar> From: CCP4 bulletin board on behalf of Lumbini Yadav Sent: Wednesday, July 10, 2019 13:12 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue Thank you for the valuable input On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S. And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or... But that still doesnt explain WHAT it is . Sorry not to be more help.. Eleanor On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below . I do not seen density near cysteine that was visible in Fo-Fc map On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson mailto:eleanor.dod...@york.ac.uk>> wrote: The key word for refmac is ANOM MAPONLY Are you using GUI2? Eleanor On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav mailto:lumbin...@gmail.com>> wrote: I have soaked my crystals in sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel mailto:dbon...@som.umaryland.edu>> wrote: Have you mass-speced the protein before crystallization to make sure it wasn't derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine (3-letter code: SCY). Best, Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Lumbini Yadav Sent: Tuesday, July 09, 2019 5:22 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Fo-Fc density close to cysteine residue Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma. Does anyone have an idea about what this density could be? Covalent modification? Thanks. Kind regards, Lumbini To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Thank you for the valuable input On Wed, Jul 10, 2019 at 4:25 PM Eleanor Dodson wrote: > Well - that more or less proves the residue is a CYS - there is a peak in > the PHAN map right on the S. > And that the extra density does not contain an anomalous scatterer so is > probably not a metal or a sulphate or... > > But that still doesnt explain WHAT it is . Sorry not to be more help.. > > Eleanor > > On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav wrote: > >> No I am using ccp4i. I tried doing SAD refinement in refmac and the >> output image is attached below . >> I do not seen density near cysteine that was visible in Fo-Fc map >> >> On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson >> wrote: >> >>> The key word for refmac is ANOM MAPONLY >>> Are you using GUI2? >>> Eleanor >>> >>> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav wrote: >>> >>>> I have soaked my crystals in sodium dithionite a reducing agent. I >>>> have not done mass spec but sequence is confirmed >>>> >>>> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel < >>>> dbon...@som.umaryland.edu> wrote: >>>> >>>>> Have you mass-speced the protein before crystallization to make sure >>>>> it wasn’t derivatized during expression and/or purification, or compared >>>>> the mass spec of the crystals verses purified protein? Any fancy reagents >>>>> or other reductants used during purification? >>>>> >>>>> What about S-Acetyl-cysteine (3-letter code: SCY). >>>>> >>>>> >>>>> >>>>> Best, >>>>> >>>>> >>>>> >>>>> Dan >>>>> >>>>> >>>>> >>>>> Daniel A Bonsor PhD. >>>>> >>>>> Sundberg Lab >>>>> >>>>> Institute of Human Virology >>>>> >>>>> University of Maryland, Baltimore >>>>> >>>>> 725 W Lombard Street N370 >>>>> >>>>> Baltimore >>>>> >>>>> Maryland >>>>> >>>>> MD 21201 >>>>> >>>>> Tel: (410) 706-7457 >>>>> >>>>> >>>>> >>>>> >>>>> >>>>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf >>>>> Of *Lumbini Yadav >>>>> *Sent:* Tuesday, July 09, 2019 5:22 AM >>>>> *To:* CCP4BB@JISCMAIL.AC.UK >>>>> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue >>>>> >>>>> >>>>> >>>>> Dear all, >>>>> >>>>> >>>>> >>>>> We have found a huge Fo-Fc density close to cysteine residue (see >>>>> attached image) in the structure with resolution of 1.2A. In the >>>>> crystallization condition, we have PEG 3350, Potassium phosphate >>>>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >>>>> the crystals were soaked in mother liquor containing sodium dithionite. >>>>> >>>>> >>>>> >>>>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, >>>>> CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. >>>>> But in all the screenings we do see some part of Fo-Fc density >>>>> unaddressed at 3 sigma. >>>>> >>>>> >>>>> >>>>> Does anyone have an idea about what this density could be? Covalent >>>>> modification? >>>>> >>>>> >>>>> >>>>> Thanks. >>>>> >>>>> >>>>> >>>>> >>>>> >>>>> Kind regards, >>>>> >>>>> Lumbini >>>>> >>>>> >>>>> -- >>>>> >>>>> To unsubscribe from the CCP4BB list, click the following link: >>>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>>>> >>>> >>>> -- >>>> >>>> To unsubscribe from the CCP4BB list, click the following link: >>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>>> >>> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S. And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or... But that still doesnt explain WHAT it is . Sorry not to be more help.. Eleanor On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav wrote: > No I am using ccp4i. I tried doing SAD refinement in refmac and the output > image is attached below . > I do not seen density near cysteine that was visible in Fo-Fc map > > On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson > wrote: > >> The key word for refmac is ANOM MAPONLY >> Are you using GUI2? >> Eleanor >> >> On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav wrote: >> >>> I have soaked my crystals in sodium dithionite a reducing agent. I have >>> not done mass spec but sequence is confirmed >>> >>> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel >>> wrote: >>> >>>> Have you mass-speced the protein before crystallization to make sure it >>>> wasn’t derivatized during expression and/or purification, or compared the >>>> mass spec of the crystals verses purified protein? Any fancy reagents or >>>> other reductants used during purification? >>>> >>>> What about S-Acetyl-cysteine (3-letter code: SCY). >>>> >>>> >>>> >>>> Best, >>>> >>>> >>>> >>>> Dan >>>> >>>> >>>> >>>> Daniel A Bonsor PhD. >>>> >>>> Sundberg Lab >>>> >>>> Institute of Human Virology >>>> >>>> University of Maryland, Baltimore >>>> >>>> 725 W Lombard Street N370 >>>> >>>> Baltimore >>>> >>>> Maryland >>>> >>>> MD 21201 >>>> >>>> Tel: (410) 706-7457 >>>> >>>> >>>> >>>> >>>> >>>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf >>>> Of *Lumbini Yadav >>>> *Sent:* Tuesday, July 09, 2019 5:22 AM >>>> *To:* CCP4BB@JISCMAIL.AC.UK >>>> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue >>>> >>>> >>>> >>>> Dear all, >>>> >>>> >>>> >>>> We have found a huge Fo-Fc density close to cysteine residue (see >>>> attached image) in the structure with resolution of 1.2A. In the >>>> crystallization condition, we have PEG 3350, Potassium phosphate >>>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >>>> the crystals were soaked in mother liquor containing sodium dithionite. >>>> >>>> >>>> >>>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, >>>> CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. >>>> But in all the screenings we do see some part of Fo-Fc density >>>> unaddressed at 3 sigma. >>>> >>>> >>>> >>>> Does anyone have an idea about what this density could be? Covalent >>>> modification? >>>> >>>> >>>> >>>> Thanks. >>>> >>>> >>>> >>>> >>>> >>>> Kind regards, >>>> >>>> Lumbini >>>> >>>> >>>> -- >>>> >>>> To unsubscribe from the CCP4BB list, click the following link: >>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>>> >>> >>> -- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>> >> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below . I do not seen density near cysteine that was visible in Fo-Fc map On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson wrote: > The key word for refmac is ANOM MAPONLY > Are you using GUI2? > Eleanor > > On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav wrote: > >> I have soaked my crystals in sodium dithionite a reducing agent. I have >> not done mass spec but sequence is confirmed >> >> On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel >> wrote: >> >>> Have you mass-speced the protein before crystallization to make sure it >>> wasn’t derivatized during expression and/or purification, or compared the >>> mass spec of the crystals verses purified protein? Any fancy reagents or >>> other reductants used during purification? >>> >>> What about S-Acetyl-cysteine (3-letter code: SCY). >>> >>> >>> >>> Best, >>> >>> >>> >>> Dan >>> >>> >>> >>> Daniel A Bonsor PhD. >>> >>> Sundberg Lab >>> >>> Institute of Human Virology >>> >>> University of Maryland, Baltimore >>> >>> 725 W Lombard Street N370 >>> >>> Baltimore >>> >>> Maryland >>> >>> MD 21201 >>> >>> Tel: (410) 706-7457 >>> >>> >>> >>> >>> >>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf >>> Of *Lumbini Yadav >>> *Sent:* Tuesday, July 09, 2019 5:22 AM >>> *To:* CCP4BB@JISCMAIL.AC.UK >>> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue >>> >>> >>> >>> Dear all, >>> >>> >>> >>> We have found a huge Fo-Fc density close to cysteine residue (see >>> attached image) in the structure with resolution of 1.2A. In the >>> crystallization condition, we have PEG 3350, Potassium phosphate >>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >>> the crystals were soaked in mother liquor containing sodium dithionite. >>> >>> >>> >>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, >>> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in >>> all the screenings we do see some part of Fo-Fc density unaddressed at >>> 3 sigma. >>> >>> >>> >>> Does anyone have an idea about what this density could be? Covalent >>> modification? >>> >>> >>> >>> Thanks. >>> >>> >>> >>> >>> >>> Kind regards, >>> >>> Lumbini >>> >>> >>> -- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
The key word for refmac is ANOM MAPONLY Are you using GUI2? Eleanor On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav wrote: > I have soaked my crystals in sodium dithionite a reducing agent. I have > not done mass spec but sequence is confirmed > > On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel > wrote: > >> Have you mass-speced the protein before crystallization to make sure it >> wasn’t derivatized during expression and/or purification, or compared the >> mass spec of the crystals verses purified protein? Any fancy reagents or >> other reductants used during purification? >> >> What about S-Acetyl-cysteine (3-letter code: SCY). >> >> >> >> Best, >> >> >> >> Dan >> >> >> >> Daniel A Bonsor PhD. >> >> Sundberg Lab >> >> Institute of Human Virology >> >> University of Maryland, Baltimore >> >> 725 W Lombard Street N370 >> >> Baltimore >> >> Maryland >> >> MD 21201 >> >> Tel: (410) 706-7457 >> >> >> >> >> >> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of >> *Lumbini Yadav >> *Sent:* Tuesday, July 09, 2019 5:22 AM >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue >> >> >> >> Dear all, >> >> >> >> We have found a huge Fo-Fc density close to cysteine residue (see >> attached image) in the structure with resolution of 1.2A. In the >> crystallization condition, we have PEG 3350, Potassium phosphate >> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >> the crystals were soaked in mother liquor containing sodium dithionite. >> >> >> >> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, >> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in >> all the screenings we do see some part of Fo-Fc density unaddressed at 3 >> sigma. >> >> >> >> Does anyone have an idea about what this density could be? Covalent >> modification? >> >> >> >> Thanks. >> >> >> >> >> >> Kind regards, >> >> Lumbini >> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
I have soaked my crystals in sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel wrote: > Have you mass-speced the protein before crystallization to make sure it > wasn’t derivatized during expression and/or purification, or compared the > mass spec of the crystals verses purified protein? Any fancy reagents or > other reductants used during purification? > > What about S-Acetyl-cysteine (3-letter code: SCY). > > > > Best, > > > > Dan > > > > Daniel A Bonsor PhD. > > Sundberg Lab > > Institute of Human Virology > > University of Maryland, Baltimore > > 725 W Lombard Street N370 > > Baltimore > > Maryland > > MD 21201 > > Tel: (410) 706-7457 > > > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Lumbini > Yadav > *Sent:* Tuesday, July 09, 2019 5:22 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Fo-Fc density close to cysteine residue > > > > Dear all, > > > > We have found a huge Fo-Fc density close to cysteine residue (see attached > image) in the structure with resolution of 1.2A. In the crystallization > condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and > protein was in Tris and NaCl. Before freezing the crystals were soaked in > mother liquor containing sodium dithionite. > > > > I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, > CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in > all the screenings we do see some part of Fo-Fc density unaddressed at 3 > sigma. > > > > Does anyone have an idea about what this density could be? Covalent > modification? > > > > Thanks. > > > > > > Kind regards, > > Lumbini > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Lumbini, 2.5A is close to the coordination bond length of sulfur-metal. Did you use IMAC during purification? ср, 10 июл. 2019 г. в 08:44, Eleanor Dodson < 176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>: > I am serious about checking the anomalous map!! > (trivial from REFMAC - key word anom map on) > Then do a peak search and just check first that the rogue residue IS CYS > first - you should see the S as a peak..) > > Eleanor > > > On Wed, 10 Jul 2019 at 07:38, Lumbini Yadav wrote: > >> Thanks for the reply. >> The distance between sulphur and centre of Fo-Fc map is around 2.5A. >> The density does not appear to be of hydrogen (attached image) anisotropy >> refinement also does not help. >> >> On Tue, Jul 9, 2019 at 5:13 PM Anthony Addlagatta >> wrote: >> >>> Lumbini, >>> >>> It would useful to know the distance between the sulfur and center of >>> the Fo-Fc map that you have shown. Since you have very high resolution >>> data, the extra density could be for a hydrogen atom in CSO-H (sufeneic >>> acid) or anisotropy of the oxygen atom. >>> >>> Anthony >>> ------ >>> *From: *176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk >>> *To: *CCP4BB@JISCMAIL.AC.UK >>> *Sent: *Tuesday, July 9, 2019 4:48:32 PM >>> *Subject: *Re: [ccp4bb] Fo-Fc density close to cysteine residue >>> >>> Any anomalous diffraction? >>> >>> On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav wrote: >>> >>>> Dear all, >>>> >>>> >>>> >>>> We have found a huge Fo-Fc density close to cysteine residue (see >>>> attached image) in the structure with resolution of 1.2A. In the >>>> crystallization condition, we have PEG 3350, Potassium phosphate >>>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >>>> the crystals were soaked in mother liquor containing sodium dithionite. >>>> >>>> >>>> >>>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, >>>> CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. >>>> But in all the screenings we do see some part of Fo-Fc density >>>> unaddressed at 3 sigma. >>>> >>>> >>>> >>>> Does anyone have an idea about what this density could be? Covalent >>>> modification? >>>> >>>> >>>> >>>> Thanks. >>>> >>>> >>>> >>>> Kind regards, >>>> >>>> Lumbini >>>> >>>> -- >>>> >>>> To unsubscribe from the CCP4BB list, click the following link: >>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>>> >>> >>> -- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>> >>> >>> >>> >>> -- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- Evgenii Osipov Laboratory for Biocrystallography, Department of Pharmaceutical Sciences, KU Leuven O To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
I am serious about checking the anomalous map!! (trivial from REFMAC - key word anom map on) Then do a peak search and just check first that the rogue residue IS CYS first - you should see the S as a peak..) Eleanor On Wed, 10 Jul 2019 at 07:38, Lumbini Yadav wrote: > Thanks for the reply. > The distance between sulphur and centre of Fo-Fc map is around 2.5A. The > density does not appear to be of hydrogen (attached image) anisotropy > refinement also does not help. > > On Tue, Jul 9, 2019 at 5:13 PM Anthony Addlagatta > wrote: > >> Lumbini, >> >> It would useful to know the distance between the sulfur and center of the >> Fo-Fc map that you have shown. Since you have very high resolution data, >> the extra density could be for a hydrogen atom in CSO-H (sufeneic acid) or >> anisotropy of the oxygen atom. >> >> Anthony >> -- >> *From: *176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk >> *To: *CCP4BB@JISCMAIL.AC.UK >> *Sent: *Tuesday, July 9, 2019 4:48:32 PM >> *Subject: *Re: [ccp4bb] Fo-Fc density close to cysteine residue >> >> Any anomalous diffraction? >> >> On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav wrote: >> >>> Dear all, >>> >>> >>> >>> We have found a huge Fo-Fc density close to cysteine residue (see >>> attached image) in the structure with resolution of 1.2A. In the >>> crystallization condition, we have PEG 3350, Potassium phosphate >>> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >>> the crystals were soaked in mother liquor containing sodium dithionite. >>> >>> >>> >>> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, >>> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in >>> all the screenings we do see some part of Fo-Fc density unaddressed at >>> 3 sigma. >>> >>> >>> >>> Does anyone have an idea about what this density could be? Covalent >>> modification? >>> >>> >>> >>> Thanks. >>> >>> >>> >>> Kind regards, >>> >>> Lumbini >>> >>> -- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >>> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> >> >> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Thanks for the reply. The distance between sulphur and centre of Fo-Fc map is around 2.5A. The density does not appear to be of hydrogen (attached image) anisotropy refinement also does not help. On Tue, Jul 9, 2019 at 5:13 PM Anthony Addlagatta wrote: > Lumbini, > > It would useful to know the distance between the sulfur and center of the > Fo-Fc map that you have shown. Since you have very high resolution data, > the extra density could be for a hydrogen atom in CSO-H (sufeneic acid) or > anisotropy of the oxygen atom. > > Anthony > -- > *From: *176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk > *To: *CCP4BB@JISCMAIL.AC.UK > *Sent: *Tuesday, July 9, 2019 4:48:32 PM > *Subject: *Re: [ccp4bb] Fo-Fc density close to cysteine residue > > Any anomalous diffraction? > > On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav wrote: > >> Dear all, >> >> >> >> We have found a huge Fo-Fc density close to cysteine residue (see >> attached image) in the structure with resolution of 1.2A. In the >> crystallization condition, we have PEG 3350, Potassium phosphate >> monobasic, glycerol and protein was in Tris and NaCl. Before freezing >> the crystals were soaked in mother liquor containing sodium dithionite. >> >> >> >> I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, >> CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in >> all the screenings we do see some part of Fo-Fc density unaddressed at 3 >> sigma. >> >> >> >> Does anyone have an idea about what this density could be? Covalent >> modification? >> >> >> >> Thanks. >> >> >> >> Kind regards, >> >> Lumbini >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Hard to see from a static image, but could it be an alternative conformation? Pavel On Tue, Jul 9, 2019 at 2:32 AM Lumbini Yadav wrote: > Dear all, > > > > We have found a huge Fo-Fc density close to cysteine residue (see attached > image) in the structure with resolution of 1.2A. In the crystallization > condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and > protein was in Tris and NaCl. Before freezing the crystals were soaked in > mother liquor containing sodium dithionite. > > > > I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, > CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in > all the screenings we do see some part of Fo-Fc density unaddressed at 3 > sigma. > > > > Does anyone have an idea about what this density could be? Covalent > modification? > > > > Thanks. > > > > Kind regards, > > Lumbini > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Refining B-aniso's will clean up a difference map at that resolution.
Sent from Yahoo Mail on Android
On Tue, 9 Jul 2019 at 16:57, Bonsor, Daniel wrote:
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Have you mass-speced the protein before crystallization to make sure it wasn’t
derivatized during expression and/or purification, or compared the mass spec of
the crystals verses purified protein? Any fancy reagents or other reductants
used during purification?
What about S-Acetyl-cysteine (3-letter code: SCY).
Best,
Dan
Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK]On Behalf Of Lumbini
Yadav
Sent: Tuesday, July 09, 2019 5:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fo-Fc density close to cysteine residue
Dear all,
We have found a huge Fo-Fc density close to cysteine residue (see attached
image) in the structure with resolution of 1.2A. In the crystallization
condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and
protein was in Tris and NaCl. Before freezing the crystals were soaked in
mother liquor containing sodium dithionite.
I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD,
CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the
screenings we do see some part of Fo-Fc density unaddressed at 3 sigma.
Does anyone have an idea about what this density could be? Covalent
modification?
Thanks.
Kind regards,
Lumbini
To unsubscribe from the CCP4BB list, click the following link:
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To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Fo-Fc density close to cysteine residue
Have you mass-speced the protein before crystallization to make sure it wasn’t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine (3-letter code: SCY). Best, Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Lumbini Yadav Sent: Tuesday, July 09, 2019 5:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fo-Fc density close to cysteine residue Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma. Does anyone have an idea about what this density could be? Covalent modification? Thanks. Kind regards, Lumbini To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Lumbini, It would useful to know the distance between the sulfur and center of the Fo-Fc map that you have shown. Since you have very high resolution data, the extra density could be for a hydrogen atom in CSO-H (sufeneic acid) or anisotropy of the oxygen atom. Anthony From: 176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 9, 2019 4:48:32 PM Subject: Re: [ccp4bb] Fo-Fc density close to cysteine residue Any anomalous diffraction? On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav < [ mailto:lumbin...@gmail.com | lumbin...@gmail.com ] > wrote: Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO 3 , SO 2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma. Does anyone have an idea about what this density could be? Covalent modification? Thanks. Kind regards, Lumbini To unsubscribe from the CCP4BB list, click the following link: [ https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 | https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 ] To unsubscribe from the CCP4BB list, click the following link: [ https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 | https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 ] To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fo-Fc density close to cysteine residue
Any anomalous diffraction? On Tue, 9 Jul 2019 at 10:32, Lumbini Yadav wrote: > Dear all, > > > > We have found a huge Fo-Fc density close to cysteine residue (see attached > image) in the structure with resolution of 1.2A. In the crystallization > condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and > protein was in Tris and NaCl. Before freezing the crystals were soaked in > mother liquor containing sodium dithionite. > > > > I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, > CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in > all the screenings we do see some part of Fo-Fc density unaddressed at 3 > sigma. > > > > Does anyone have an idea about what this density could be? Covalent > modification? > > > > Thanks. > > > > Kind regards, > > Lumbini > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Fo-Fc density close to cysteine residue
Dear all, We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite. I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma. Does anyone have an idea about what this density could be? Covalent modification? Thanks. Kind regards, Lumbini To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
