Re: [ccp4bb] Phaser 2.8.3: Hendrickson-Lattman coefficients generated from dataset lacking anomalous signal
Dear sir Thank you for clearing that. I checked back to see that HLC/D are invariably 0 for all reflections, with the non-zero HLA/B supposedly having been originated from the probability distribution of phases *calculated* by phaser. Hopefully, I have not misunderstood it. Thanks and regards Nitin Kulhar On Thu, Feb 8, 2024 at 9:07 PM Randy John Read wrote: > Hi, > > Hendrickson-Lattman coefficients are just a way of storing phase > probability information, and they can come from different sources including > atomic models. Phaser puts in HL coefficients because they could be handy > under some circumstances for combining the phase information from > experimental phasing. You might notice that only A and B are non-zero for > the molecular replacement HL coefficients. That’s because the phase > probability distribution is unimodal for calculated phases, whereas it’s > generally bimodal for experimental phases (thus requiring more > coefficients). > > Best wishes, > > Randy Read > > > On 8 Feb 2024, at 14:32, Nitin Kulhar < > 9dfccc771c91-dmarc-requ...@jiscmail.ac.uk> wrote: > > > > Dear all > > > > Is anomalous diffraction necessary for determining experimental phases > and the Hendrickson-Lattman coefficients (HLA, HLB, HLC, and HLD)? > > > > MR solution from Phaser 2.8.3 (interfaced in ccp4 8.0.000 suite) seems > to be generating HLA/B/C/D coefficients from an x-ray diffraction dataset. > > > > Wavelength: > > 1.54179 Angstrom. > > > > Sample: > > • > > Protein-small molecule ligand complex crystal. > > • No anomalous scatterer in the protein, the ligand, or the > crystallization condition. > > > > Data reduction: > > Xtriage and aimless analyses did not indicate significant anomalous > signal. > > > > I would appreciate any help in understanding the reasons for these > observations. > > > > Thanks and regards > > Nitin Kulhar > > PhD student > > c/o Dr Rajakumara Eerappa > > Macromolecular Structural Biology Lab > > Department of Biotechnology > > Indian Institute of Technology Hyderabad > > Kandi, Sangareddy > > Telangana, India - 502284 > > > > 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 > > - > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: +44 1223 336500 > The Keith Peters Building > Hills Road E-mail: > rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > -- 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/
[ccp4bb] Phaser 2.8.3: Hendrickson-Lattman coefficients generated from dataset lacking anomalous signal
Dear all Is anomalous diffraction necessary for determining experimental phases and the Hendrickson-Lattman coefficients (HLA, HLB, HLC, and HLD)? MR solution from Phaser 2.8.3 (interfaced in ccp4 8.0.000 suite) seems to be generating HLA/B/C/D coefficients from an x-ray diffraction dataset. Wavelength: 1.54179 Angstrom. Sample: 1. Protein-small molecule ligand complex crystal. 2. No anomalous scatterer in the protein, the ligand, or the crystallization condition. Data reduction: Xtriage and aimless analyses did not indicate significant anomalous signal. I would appreciate any help in understanding the reasons for these observations. Thanks and regards Nitin Kulhar PhD student c/o Dr Rajakumara Eerappa Macromolecular Structural Biology Lab Department of Biotechnology Indian Institute of Technology Hyderabad Kandi, Sangareddy Telangana, India - 502284 -- 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] Intractable outliers in wwPDB validation report
Hello everyone. Many thanks for the thoughts on the matter. Dear Dr Abhinav You can try to run a simulated annealing in CNS and then try to refine that > model and see if that solves the problem. This would most likely help in > getting rid of the overfitting problems. > I have trouble installing CNS on my machine which has Windows OS. Nevertheless, rigid body refinement (RBR) and simulated annealing (SA) (and combinations thereof) with phenix led to distant Rf and Rw (a sign of model bias) at the beginning of refinement and even more so towards the end (*at tedium*). Toggling off of RBR and SA, however, bridged the gap between Rf and Rw while lowering them at the same time. Dear Dr Mark The R/Rfree values seem very low for a 2.67Å resolution structure. Perhaps > it was over-refined? Difficult to know without looking at the specific case > and details of the data quality though. > PFA merging stats. Asymmetric unit seems to have 8 molecules @~44% bulk solvent content. I can only speculate as to systematic problems arising in intensity correction accompanying the MR run on account of a combination of NCS (~26% off-origin peak in Patterson map) and a screw axis symmetry (P 1 21 1 being the space group). The best way to resolve validation concerns is to inspect the outliers > individually - it might be worth getting a second pair of experienced eyes > to look at them. Perhaps there is a peptide flip or a cis-peptide that you > missed. > Finally, it is not usually possible to remove all validation concerns, > especially at intermediate resolution like 2.67Å. > Among the clashes and the other geometry outliers, there are cis-prolines in the final model - same as the MR model that was pulled from RCSB. As such, I decided to leave them in. Trying to fix them in coot serves no good, as they come undone upon uploading the resultant structure to wwPDB. Over-fitting, as you pointed out, was also a result of the umpteen attempts to iron these issues out with refinement/remodelling. Dear Professor Eleanor I use the validation report to check for obvious modelling errors but if > you can’t find any, you have to just submit the results of your > experiment... > I have sent a message to wwPDB curator requesting to admit the current model - with outliers. Response awaited. Dear Dr Powell I will submit the data to PDB-REDO Dear Professor Cooper Many outliers have been done away with. The ones that remain seem truly intractable. We have been trying to submit the structure in its current state barring only the red exclamation point on the validation reports section, even with green ticks on every other section. Thank you all for the helpful thoughts, again. Please revert for further information. Yours faithfully Nitin Kulhar On Mon, Sep 25, 2023 at 5:44 PM Jon Cooper wrote: > Hello, unless it's changed over the last couple of years, the pdb rightly > used to let you deposit a structure with outliers, hopefully not too many, > though ;-0 > > I take it the stalling you reported probably comes from whoever is looking > over your shoulder. > > Best wishes, Jon Cooper. jon.b.coo...@protonmail.com > > Sent from Proton Mail mobile > > > > Original Message > On 23 Sept 2023, 02:29, Nitin Kulhar < > 9dfccc771c91-dmarc-requ...@jiscmail.ac.uk> wrote: > > > Dear all > > We have refined (Refmac5) a crystallographic structure with Rw/Rf values > 0.19/0.22 (Resln 2.67). However, the deposition has stalled on account of > the wwPDB's preliminary validation report, which indicates map/model and > geometry issues, with each criterion containing a few instances. We tried > to correct these by varying overall geometry restraint weights, e.g. > decreasing overall weights from default value of 1.0, incementally > decreasing sigmas corresponding to the planarity restraint term. This did > not resolve the issues. > > In another approach, real space refinement by hand (against 2fo-fc) in > coot brought the geometry parameters within acceptable limits in addition > to improved apparent agreement with electron density (2fo-fc, sigma=1), but > uploading the resultant coordinates seems to undo the changes made in coot, > as indicated by reappearance of same outliers in the subsequent validation > report. > > I request your kind suggestions in this regard. Please also revert for any > further information. > > Thanks > Nitin Kulhar > PhD student > c/o Dr Rajakumara Eerappa > Macromolecular Structural Biology Group > Department of Biotechnology > Indian Institute of Technology Hyderabad > Kandi, Sangareddy > Telangana, India 502284 > > 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. > > -- > &
[ccp4bb] Intractable outliers in wwPDB validation report
Dear all We have refined (Refmac5) a crystallographic structure with Rw/Rf values 0.19/0.22 (Resln 2.67). However, the deposition has stalled on account of the wwPDB's preliminary validation report, which indicates map/model and geometry issues, with each criterion containing a few instances. We tried to correct these by varying overall geometry restraint weights, e.g. decreasing overall weights from default value of 1.0, incementally decreasing sigmas corresponding to the planarity restraint term. This did not resolve the issues. In another approach, real space refinement by hand (against 2fo-fc) in coot brought the geometry parameters within acceptable limits in addition to improved apparent agreement with electron density (2fo-fc, sigma=1), but uploading the resultant coordinates seems to undo the changes made in coot, as indicated by reappearance of same outliers in the subsequent validation report. I request your kind suggestions in this regard. Please also revert for any further information. Thanks Nitin Kulhar PhD student c/o Dr Rajakumara Eerappa Macromolecular Structural Biology Group Department of Biotechnology Indian Institute of Technology Hyderabad Kandi, Sangareddy Telangana, India 502284 -- 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
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/
[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/
