Re: [ccp4bb] [EXTERNAL]Re: [ccp4bb] Modeling Disulfide Bond Occupancies
Thank you so much From: Eleanor Dodson Date: Monday, May 6, 2024 at 3:30 PM To: Liliana Margent Cc: CCP4BB@jiscmail.ac.uk Subject: [EXTERNAL]Re: [ccp4bb] Modeling Disulfide Bond Occupancies ***ATTENTION: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.*** Well - I turn off occupancy refinement once there is a sort of consensus.. Disulphides often break after long exposures and you see the positive and negative blobs clearly. I must admit I usually just set 0.5 as occ , fix the surviving disulphide link and let the B values suggest a better ratio than o.5:0.5.. Eleanor On Mon, 6 May 2024 at 20:20, Liliana Margent mailto:lmarg...@gradcenter.cuny.edu>> wrote: Greetings everyone, I'm currently in the process of modeling a disulfide bond in two structures. However, when I attempt to model single occupancy for the cysteines involved in the bond, negative density blobs emerge within the disulfide bond. This suggests the possibility of alternate conformations for the cysteines. Yet, when I endeavor to model alternate conformations for both cysteines, their occupancies do not align despite running refinement with a bond parameter file that specifies the link. To illustrate, I initiate the refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s bond. Has anyone else encountered this issue before, or does anyone have suggestions on how to refine these cysteines to achieve coherent occupancies? Thank you for any insights you can provide. Best, Liliana To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk/>, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Modeling Disulfide Bond Occupancies
Do not know what the resolution is here, try an anisotropic refinement of the Cys side chains first to see if this solves your problem. __ Dr. math. et dis. nat. Jeroen R. Mesters University of Lübeck https://orcid.org/-0001-8532-6699 Am 06.05.2024 um 21:48 schrieb Eleanor Dodson <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>: But are there two conformations of the disulphide? or one disulphide and one broken link? Eleaor On Mon, 6 May 2024 at 20:42, Dr. Kevin M Jude mailto:kj...@stanford.edu>> wrote: I have done this in shelxl or phenix refinement, you can define occupancy groups (or free variables in shelxl) so that 472A and 384A are one group, 472B and 384B are another. Pretty sure there is a similar solution in refmac. Though also if the 384A rotamer doesn’t clash with the 472B rotamer, you could have three states A/A (disulfide), A/B (no disulfide), B/B (no disulfide). -- Kevin Jude, PhD Structural Biology Research Specialist, Garcia Lab Howard Hughes Medical Institute Stanford University School of Medicine Beckman B177, 279 Campus Drive, Stanford CA 94305 From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Liliana Margent mailto:lmarg...@gradcenter.cuny.edu>> Date: Monday, May 6, 2024 at 12:20 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: [ccp4bb] Modeling Disulfide Bond Occupancies Greetings everyone, I'm currently in the process of modeling a disulfide bond in two structures. However, when I attempt to model single occupancy for the cysteines involved in the bond, negative density blobs emerge within the disulfide bond. This suggests the possibility of alternate conformations for the cysteines. Yet, when I endeavor to model alternate conformations for both cysteines, their occupancies do not align despite running refinement with a bond parameter file that specifies the link. To illustrate, I initiate the refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s bond. Has anyone else encountered this issue before, or does anyone have suggestions on how to refine these cysteines to achieve coherent occupancies? Thank you for any insights you can provide. Best, Liliana To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk/>, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Modeling Disulfide Bond Occupancies
In the CCP4 program refmac5, you specify occupancy groups and then specify how to refine them. The setup, afaik, is not automatic. Documentation for how to do it is here: http://www.ysbl.york.ac.uk/refmac/data/refmac_keywords.html#Occupancy You may also find this link helpful? https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg51707.html -James Holton MAD Scientist On 5/6/2024 12:19 PM, Liliana Margent wrote: Greetings everyone, I'm currently in the process of modeling a disulfide bond in two structures. However, when I attempt to model single occupancy for the cysteines involved in the bond, negative density blobs emerge within the disulfide bond. This suggests the possibility of alternate conformations for the cysteines. Yet, when I endeavor to model alternate conformations for both cysteines, their occupancies do not align despite running refinement with a bond parameter file that specifies the link. To illustrate, I initiate the refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s bond. Has anyone else encountered this issue before, or does anyone have suggestions on how to refine these cysteines to achieve coherent occupancies? Thank you for any insights you can provide. Best, Liliana To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Modeling Disulfide Bond Occupancies
But are there two conformations of the disulphide? or one disulphide and one broken link? Eleaor On Mon, 6 May 2024 at 20:42, Dr. Kevin M Jude wrote: > I have done this in shelxl or phenix refinement, you can define occupancy > groups (or free variables in shelxl) so that 472A and 384A are one group, > 472B and 384B are another. Pretty sure there is a similar solution in > refmac. Though also if the 384A rotamer doesn’t clash with the 472B > rotamer, you could have three states A/A (disulfide), A/B (no disulfide), > B/B (no disulfide). > > > > -- > > Kevin Jude, PhD > > Structural Biology Research Specialist, Garcia Lab > > Howard Hughes Medical Institute > > Stanford University School of Medicine > > Beckman B177, 279 Campus Drive, Stanford CA 94305 > > > > > > *From: *CCP4 bulletin board on behalf of Liliana > Margent > *Date: *Monday, May 6, 2024 at 12:20 PM > *To: *CCP4BB@JISCMAIL.AC.UK > *Subject: *[ccp4bb] Modeling Disulfide Bond Occupancies > > Greetings everyone, > > I'm currently in the process of modeling a disulfide bond in two > structures. However, when I attempt to model single occupancy for the > cysteines involved in the bond, negative density blobs emerge within the > disulfide bond. This suggests the possibility of alternate conformations > for the cysteines. > > Yet, when I endeavor to model alternate conformations for both cysteines, > their occupancies do not align despite running refinement with a bond > parameter file that specifies the link. To illustrate, I initiate the > refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as > A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 > A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s > bond. > > Has anyone else encountered this issue before, or does anyone have > suggestions on how to refine these cysteines to achieve coherent > occupancies? > > Thank you for any insights you can provide. > > Best, > Liliana > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Modeling Disulfide Bond Occupancies
I have done this in shelxl or phenix refinement, you can define occupancy groups (or free variables in shelxl) so that 472A and 384A are one group, 472B and 384B are another. Pretty sure there is a similar solution in refmac. Though also if the 384A rotamer doesn’t clash with the 472B rotamer, you could have three states A/A (disulfide), A/B (no disulfide), B/B (no disulfide). -- Kevin Jude, PhD Structural Biology Research Specialist, Garcia Lab Howard Hughes Medical Institute Stanford University School of Medicine Beckman B177, 279 Campus Drive, Stanford CA 94305 From: CCP4 bulletin board on behalf of Liliana Margent Date: Monday, May 6, 2024 at 12:20 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Modeling Disulfide Bond Occupancies Greetings everyone, I'm currently in the process of modeling a disulfide bond in two structures. However, when I attempt to model single occupancy for the cysteines involved in the bond, negative density blobs emerge within the disulfide bond. This suggests the possibility of alternate conformations for the cysteines. Yet, when I endeavor to model alternate conformations for both cysteines, their occupancies do not align despite running refinement with a bond parameter file that specifies the link. To illustrate, I initiate the refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s bond. Has anyone else encountered this issue before, or does anyone have suggestions on how to refine these cysteines to achieve coherent occupancies? Thank you for any insights you can provide. Best, Liliana To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk>, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Modeling Disulfide Bond Occupancies
Well - I turn off occupancy refinement once there is a sort of consensus.. Disulphides often break after long exposures and you see the positive and negative blobs clearly. I must admit I usually just set 0.5 as occ , fix the surviving disulphide link and let the B values suggest a better ratio than o.5:0.5.. Eleanor On Mon, 6 May 2024 at 20:20, Liliana Margent wrote: > Greetings everyone, > > I'm currently in the process of modeling a disulfide bond in two > structures. However, when I attempt to model single occupancy for the > cysteines involved in the bond, negative density blobs emerge within the > disulfide bond. This suggests the possibility of alternate conformations > for the cysteines. > > Yet, when I endeavor to model alternate conformations for both cysteines, > their occupancies do not align despite running refinement with a bond > parameter file that specifies the link. To illustrate, I initiate the > refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as > A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 > A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s > bond. > > Has anyone else encountered this issue before, or does anyone have > suggestions on how to refine these cysteines to achieve coherent > occupancies? > > Thank you for any insights you can provide. > > Best, > Liliana > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Modeling Disulfide Bond Occupancies
Greetings everyone, I'm currently in the process of modeling a disulfide bond in two structures. However, when I attempt to model single occupancy for the cysteines involved in the bond, negative density blobs emerge within the disulfide bond. This suggests the possibility of alternate conformations for the cysteines. Yet, when I endeavor to model alternate conformations for both cysteines, their occupancies do not align despite running refinement with a bond parameter file that specifies the link. To illustrate, I initiate the refinement with occupancies for Cys472 as A0.75/B0.25 and for C384 as A0.75/B0.25, but post-refinement, the output occupancies appear as Cys472 A0.84/B0.16 and for C384 A0.97/B0.03. Where A confs participate in the s-s bond. Has anyone else encountered this issue before, or does anyone have suggestions on how to refine these cysteines to achieve coherent occupancies? Thank you for any insights you can provide. Best, Liliana To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
