Hi Eleanor, Thinking about this you are most likely correct that COOT has created this issue. The coordinates were in PDB format.
Best wishes, Joanna ________________________________ From: Eleanor Dodson <eleanor.dod...@york.ac.uk> Sent: 03 February 2025 15:17 To: Joanna Zukowska <jz2...@bath.ac.uk> Cc: CCP4BB@jiscmail.ac.uk <CCP4BB@jiscmail.ac.uk> Subject: Re: [ccp4bb] Bonds Breaking After Refinement CAUTION: This email came from outside of the University. To keep your account safe, only click on links and open attachments if you know the person who sent the email, or you expected to receive this communication. I think it is probably a COOT issue - if you add a TERMINAL RESIDUE in COOT after res 430 say, it should delete the previous TER record.. A TER record is meant to flag the end of a protein chain, so I guess really REFMAC is to be congratulated for acknowledging that, and the other programs congratulated for using their common sense and ignoring it?? Were you working with coordinates in PDB format or mmCIF? Eleanor On Mon, 3 Feb 2025 at 15:01, Joanna Zukowska <jz2...@bath.ac.uk<mailto:jz2...@bath.ac.uk>> wrote: Unfortunately we cannot share this file due to an NDA. However we think the problems were due to some missing residues being built in through the "get monomer" option which COOT and molecular replacement (PHASER) does not seem to have a problem with, but for some reason REFMAC does. Molecular replacement seems to have corrected any of the .pdb problems which REFMAC could not read, and any refinement steps from this point seem to be working fine. Molecular replacement removed all the TER lines and corrected any numbering in the file, as well as changed all the peptide atoms to "ATOM" from "HETATM". All the best, Joanna ________________________________ From: Eleanor Dodson <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> Sent: 03 February 2025 14:50 To: Joanna Zukowska <jz2...@bath.ac.uk<mailto:jz2...@bath.ac.uk>> Cc: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk> <CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>> Subject: Re: [ccp4bb] Bonds Breaking After Refinement You don't often get email from eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>. Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> CAUTION: This email came from outside of the University. To keep your account safe, only click on links and open attachments if you know the person who sent the email, or you expected to receive this communication. Could you send the rogue coordinate file or is it confidential? it might perhaps be valuable to find WHY it caused such problems.. On Mon, 3 Feb 2025 at 14:22, Joanna Zukowska <000100b050ebf83d-dmarc-requ...@jiscmail.ac.uk<mailto:000100b050ebf83d-dmarc-requ...@jiscmail.ac.uk>> wrote: Hi all, Thank you for all your advice. We ended up re-running the coordinate file through molecular replacement and it seems to have corrected all the problems within the file mentioned. All the best, Joanna ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Keitaro Yamashita <0000eb4171b56fc1-dmarc-requ...@jiscmail.ac.uk<mailto:0000eb4171b56fc1-dmarc-requ...@jiscmail.ac.uk>> Sent: 03 February 2025 12:55 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] Bonds Breaking After Refinement [You don't often get email from 0000eb4171b56fc1-dmarc-requ...@jiscmail.ac.uk<mailto:0000eb4171b56fc1-dmarc-requ...@jiscmail.ac.uk>. Learn why this is important at https://aka.ms/LearnAboutSenderIdentification ] CAUTION: This email came from outside of the University. To keep your account safe, only click on links and open attachments if you know the person who sent the email, or you expected to receive this communication. Dear Eleanor, > From log: refmac thinks there are 433 (A) or 426 (B) residues > but the VDW restraints seem to involve higher number residues . (of course > you may not have numbered things from 1...) > But why does it think the ligands include peptides?? This is because gemmi (which generates restraints within the refmacat scheme) interpreted everything after residue 430 of chain A as ligand, presumably due to an incorrect TER card. H2/H3 atoms (attached to N atoms in the main chain) were also generated incorrectly because these residues were treated as "ligand" (outside the polymer; otherwise, only "H" atom should be generated). Best regards, Keitaro On Mon, 3 Feb 2025 at 21:39, Eleanor Dodson <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> wrote: > > Hmmm = Keitaro is right - there is something odd for the last few residues.. > I would look carefully at the input coordinates about 430 > > From log: refmac thinks there are 433 (A) or 426 (B) residues > but the VDW restraints seem to involve higher number residues . (of course > you may not have numbered things from 1...) > But why does it think the ligands include peptides?? > > And why does REFMAC think this?? 25 chains?? Each of the last few residues > must be seen as a separate chain.. > Noted: A + B + C +14+8 = 25 > > ------------------------------------------ > Number of chains : 25 > > Thinking this is a bad VDW contact - A 430 CYS C . - A 431 GLN H3 - > will mean REFMAC wants to blow those residues apart.. > > Check the coordinates input to COOT and those output.. > You havent inserted an EDO or PEG with inappropriate numbering? > > > Creating restraints.. > > Chain info: > chain A > PeptideL: -2..430 (433 residues) > ligands: GLN HIS GLU ASN GLY PRO ARG EDO (14 residues) > chain B > PeptideL: -1..430 (426 residues) > gap between 306(THR) and 309(SER) > gap between 325(GLU) and 330(LYS) > ligands: PEG HIS GLN EDO (8 residues) > chain C > ligands: HOH (215 residues) > > > > **** VDW outliers > **** > > VDW deviations from the ideal >10.000Sigma or dist < 1.000 will be monitored > > A 430 CYS C . - A 431 GLN H3 . mod.= 0.582 id.= 2.950 dev= -2.37 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 431 GLN C . - A 432 ARG H3 . mod.= 0.569 id.= 2.950 dev= -2.38 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 433 PRO C . - A 434 GLY H3 . mod.= 0.547 id.= 2.950 dev= -2.40 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 434 GLY C . - A 435 GLU H3 . mod.= 0.528 id.= 2.950 dev= -2.42 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 435 GLU C . - A 436 ASN H2 . mod.= 0.673 id.= 2.950 dev= -2.28 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 447 HIS C . - A 448 HIS H3 . mod.= 0.829 id.= 2.950 dev= -2.12 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 448 HIS C . - A 449 HIS H3 . mod.= 0.489 id.= 2.950 dev= -2.46 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > A 449 HIS C . - A 450 HIS H3 . mod.= 0.849 id.= 2.950 dev= -2.10 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > .... > B 318 LYS HD3 . - B 331 PHE HE1 . mod.= 0.971 id.= 2.400 dev= -1.43 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > (This one is probably correct..) > > B 448 HIS C . - B 449 HIS H3 . mod.= 0.529 id.= 2.950 dev= -2.42 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > B 449 HIS C . - B 450 HIS H3 . mod.= 0.835 id.= 2.950 dev= -2.11 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > B 450 HIS C . - B 451 HIS H3 . mod.= 0.932 id.= 2.950 dev= -2.02 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > B 451 HIS C . - B 452 HIS H . mod.= 0.542 id.= 2.950 dev= -2.41 > sig.= 0.20 sym.= 1 0 0 0 ncs 1 type = 1 > > > > > > On Mon, 3 Feb 2025 at 11:21, Keitaro Yamashita > <0000eb4171b56fc1-dmarc-requ...@jiscmail.ac.uk<mailto:0000eb4171b56fc1-dmarc-requ...@jiscmail.ac.uk>> > wrote: >> >> Dear Joanna, >> >> There might be an unnecessary TER card in your PDB file between >> residues 430 and 431 of chain A. Could you check that? The log file >> suggests that residues after 430 are being recognised as a non-polymer >> part. >> >> Best regards, >> Keitaro >> >> On Mon, 3 Feb 2025 at 20:12, Joanna Zukowska >> <000100b050ebf83d-dmarc-requ...@jiscmail.ac.uk<mailto:000100b050ebf83d-dmarc-requ...@jiscmail.ac.uk>> >> wrote: >> > >> > Good morning Eleanor, >> > >> > Thank you for your response, I have attached the log file. As far as I am >> > aware we are not requesting unrestrained refinement. >> > >> > Best wishes, >> > Joanna >> > ________________________________ >> > From: Eleanor Dodson >> > <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> >> > Sent: 03 February 2025 10:47 >> > To: Joanna Zukowska <jz2...@bath.ac.uk<mailto:jz2...@bath.ac.uk>> >> > Cc: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk> >> > <CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>> >> > Subject: Re: [ccp4bb] Bonds Breaking After Refinement >> > >> > You don't often get email from >> > eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>. Learn why >> > this is important >> > >> > CAUTION: This email came from outside of the University. To keep your >> > account safe, only click on links and open attachments if you know the >> > person who sent the email, or you expected to receive this communication. >> > >> > >> > >> > That is VERY odd - can you send a log file? >> > And are you sure you arent accidentally requesting "unrestrained >> > refinement"? >> > In REFMAC that would be triggered by a keyword >> > REFI UNRE >> > >> > Eleanor >> > >> > >> > On Mon, 3 Feb 2025 at 09:37, Joanna Zukowska >> > <000100b050ebf83d-dmarc-requ...@jiscmail.ac.uk<mailto:000100b050ebf83d-dmarc-requ...@jiscmail.ac.uk>> >> > wrote: >> > >> > Hi all, >> > >> > Currently my colleague and I are experiencing a problem with peptide bonds >> > breaking after refinement. Has anyone else experienced this or knows how >> > to fix this problem? Selecting detect and apply covalent linkages in the >> > restraints does not help. >> > >> > Best wishes, >> > Joanna >> > >> > >> > >> > ________________________________ >> > >> > To unsubscribe from the CCP4BB list, click the following link: >> > https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=05%7C02%7Cjz2424%40BATH.AC.UK%7Cbdd13fe98f2d4578a9d608dd4452378a%7C377e3d224ea1422db0ad8fcc89406b9e%7C0%7C0%7C638741842099698647%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=cKbBIxDyZnS%2BGwHDv3rPBKuUXsMFicDdmqR2b0GDBcg%3D&reserved=0<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> >> > >> > >> > ________________________________ >> > >> > To unsubscribe from the CCP4BB list, click the following link: >> > 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