Re: [ccp4bb] NCS consideration during refinement vis-a-vis ligand occupancy and flexible loops
Thank you Dr Robbie and Prof Eleanor for clearing that up. I will incorporate the suggestion to try to improve the maps with default restraints for local NCS before applying occupancy restraints and share how that works out. Best regards. Nitin On Wed, Apr 12, 2023 at 3:29 PM Eleanor Dodson < 176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote: > As Robbie says, in such a case I just blindly refine with local NCS > restraints - this should improve the greater part of the model and thus > provide you with clearer maps. It is quite common for different copies of > the monomer to have differences - after all the crystal environment will be > different - but this should become clearer as refinement progresses. The > programs are pretty good at smudging out errors - B values usually go sky > high, and once you have a better map you can set the occupancies of the > poorly ordered bits to0.00 and see what, if anything comes back. > Eleanor > > On Wed, 12 Apr 2023 at 10:27, Robbie Joosten > wrote: > >> The fact that your protomers have different density levels does not mean >> they are structurally different. The prior assumption should be that they >> are the same unless proven otherwise. So I would keep the (local!) NCS >> restraints in the initial stages and only remove them if it becomes >> apparent that this hurts refinement. No need to worry about the density >> averaging out. The models may average out but the density should still have >> enough signal to show any real differences. >> >> HTH, >> Robbie >> >> > -Original Message- >> > From: CCP4 bulletin board On Behalf Of Nitin >> > Kulhar >> > Sent: Wednesday, April 12, 2023 10:02 >> > To: CCP4BB@JISCMAIL.AC.UK >> > Subject: [ccp4bb] NCS consideration during refinement vis-a-vis ligand >> > occupancy and flexible loops >> > >> > Hello all >> > >> > I am writing to request opinions from the community regarding the >> following: >> > >> > Situation: An ASU comprising a non-crystallographic homo-octamer of a >> > biological monomer was obtained from MR. Electron density in the >> initial 2Fo- >> > Fc, as well as Fo-Fc maps, seems to vary widely* across the eight >> protomers for >> > >> > * the supposedly co-crystallized ligand (Kd ~100 micro-molar, >> determined >> > with ITC) and >> > * 1-2 flexible loops (too far from the ligand to interact with it >> directly) >> > >> > >> > Decision: Before commencing to do refinement in such a case, would it be >> > advisable to omit the flexible loops / binding site residues from the >> NCS >> > reference group to avoid inadvertently averaging out the density of >> structural >> > elements with partial occupancies (ligands and flexible loops)? >> > >> > * varying from non-existent in some protomers to huge unmodeled blobs in >> > others. >> > >> > Please write for any clarifications / further details. I would be >> highly grateful for >> > any help in this regard. >> > >> > Best regards. >> > >> > Nitin Kulhar >> > >> > PhD student >> > c/o Dr Rajakumara Eerappa >> > Macromolecular Structural Biology Group >> > Indian Institute of Technology Hyderabad >> > Kandi, Sangareddy >> > Telangana, India - 502285 >> > >> > Disclaimer:- This footer text is to convey that this email is sent by >> one of the >> > users of IITH. So, do not mark it as SPAM. >> > >> > >> > >> > >> > 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/ >> > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > -- Disclaimer:- This footer text is to convey that this email is sent by one of the users of IITH. So, do not mark it as SPAM. 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] NCS consideration during refinement vis-a-vis ligand occupancy and flexible loops
As Robbie says, in such a case I just blindly refine with local NCS restraints - this should improve the greater part of the model and thus provide you with clearer maps. It is quite common for different copies of the monomer to have differences - after all the crystal environment will be different - but this should become clearer as refinement progresses. The programs are pretty good at smudging out errors - B values usually go sky high, and once you have a better map you can set the occupancies of the poorly ordered bits to0.00 and see what, if anything comes back. Eleanor On Wed, 12 Apr 2023 at 10:27, Robbie Joosten wrote: > The fact that your protomers have different density levels does not mean > they are structurally different. The prior assumption should be that they > are the same unless proven otherwise. So I would keep the (local!) NCS > restraints in the initial stages and only remove them if it becomes > apparent that this hurts refinement. No need to worry about the density > averaging out. The models may average out but the density should still have > enough signal to show any real differences. > > HTH, > Robbie > > > -Original Message- > > From: CCP4 bulletin board On Behalf Of Nitin > > Kulhar > > Sent: Wednesday, April 12, 2023 10:02 > > To: CCP4BB@JISCMAIL.AC.UK > > Subject: [ccp4bb] NCS consideration during refinement vis-a-vis ligand > > occupancy and flexible loops > > > > Hello all > > > > I am writing to request opinions from the community regarding the > following: > > > > Situation: An ASU comprising a non-crystallographic homo-octamer of a > > biological monomer was obtained from MR. Electron density in the initial > 2Fo- > > Fc, as well as Fo-Fc maps, seems to vary widely* across the eight > protomers for > > > > * the supposedly co-crystallized ligand (Kd ~100 micro-molar, > determined > > with ITC) and > > * 1-2 flexible loops (too far from the ligand to interact with it > directly) > > > > > > Decision: Before commencing to do refinement in such a case, would it be > > advisable to omit the flexible loops / binding site residues from the NCS > > reference group to avoid inadvertently averaging out the density of > structural > > elements with partial occupancies (ligands and flexible loops)? > > > > * varying from non-existent in some protomers to huge unmodeled blobs in > > others. > > > > Please write for any clarifications / further details. I would be highly > grateful for > > any help in this regard. > > > > Best regards. > > > > Nitin Kulhar > > > > PhD student > > c/o Dr Rajakumara Eerappa > > Macromolecular Structural Biology Group > > Indian Institute of Technology Hyderabad > > Kandi, Sangareddy > > Telangana, India - 502285 > > > > Disclaimer:- This footer text is to convey that this email is sent by > one of the > > users of IITH. So, do not mark it as SPAM. > > > > > > > > > > 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/ > 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] NCS consideration during refinement vis-a-vis ligand occupancy and flexible loops
The fact that your protomers have different density levels does not mean they are structurally different. The prior assumption should be that they are the same unless proven otherwise. So I would keep the (local!) NCS restraints in the initial stages and only remove them if it becomes apparent that this hurts refinement. No need to worry about the density averaging out. The models may average out but the density should still have enough signal to show any real differences. HTH, Robbie > -Original Message- > From: CCP4 bulletin board On Behalf Of Nitin > Kulhar > Sent: Wednesday, April 12, 2023 10:02 > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] NCS consideration during refinement vis-a-vis ligand > occupancy and flexible loops > > Hello all > > I am writing to request opinions from the community regarding the following: > > Situation: An ASU comprising a non-crystallographic homo-octamer of a > biological monomer was obtained from MR. Electron density in the initial 2Fo- > Fc, as well as Fo-Fc maps, seems to vary widely* across the eight protomers > for > > * the supposedly co-crystallized ligand (Kd ~100 micro-molar, determined > with ITC) and > * 1-2 flexible loops (too far from the ligand to interact with it > directly) > > > Decision: Before commencing to do refinement in such a case, would it be > advisable to omit the flexible loops / binding site residues from the NCS > reference group to avoid inadvertently averaging out the density of structural > elements with partial occupancies (ligands and flexible loops)? > > * varying from non-existent in some protomers to huge unmodeled blobs in > others. > > Please write for any clarifications / further details. I would be highly > grateful for > any help in this regard. > > Best regards. > > Nitin Kulhar > > PhD student > c/o Dr Rajakumara Eerappa > Macromolecular Structural Biology Group > Indian Institute of Technology Hyderabad > Kandi, Sangareddy > Telangana, India - 502285 > > Disclaimer:- This footer text is to convey that this email is sent by one of > the > users of IITH. So, do not mark it as SPAM. > > > > > 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/
[ccp4bb] NCS consideration during refinement vis-a-vis ligand occupancy and flexible loops
Hello all I am writing to request opinions from the community regarding the following: *Situation:* An ASU comprising a non-crystallographic homo-octamer of a biological monomer was obtained from MR. Electron density in the initial 2Fo-Fc, as well as Fo-Fc maps, seems to vary widely* across the eight protomers for - the supposedly co-crystallized ligand (Kd ~100 micro-molar, determined with ITC) and - 1-2 flexible loops (too far from the ligand to interact with it directly) *Decision:* Before commencing to do refinement in such a case, would it be advisable to omit the flexible loops / binding site residues from the NCS reference group to avoid inadvertently averaging out the density of structural elements with partial occupancies (ligands and flexible loops)? * varying from non-existent in some protomers to huge unmodeled blobs in others. Please write for any clarifications / further details. I would be highly grateful for any help in this regard. Best regards. Nitin Kulhar PhD student c/o Dr Rajakumara Eerappa Macromolecular Structural Biology Group Indian Institute of Technology Hyderabad Kandi, Sangareddy Telangana, India - 502285 -- Disclaimer:- This footer text is to convey that this email is sent by one of the users of IITH. So, do not mark it as SPAM. 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/
