Hi Nigel, Thanks a lot for your help! The short communication paper you directed to me helps me understand how different visualization programs deal with defined links. The geometry file (.geo) is where one should check whether or not the defined bond is linked. In fact, I found the data_link name "p" is necessary. If without supplying cif_link_GTP-G.params file to Phenix.refine, no linked bond is shown in the .geo file. With the .params file, the linked bond is found in .geo file as below: bond pdb=" O3' GTP C 1 " pdb=" P G C 2 " ideal model delta sigma weight residual 1.600 1.570 0.030 2.00e-02 2.50e+03 2.24e+00 bond pdb=" O3' GTP D 1 " pdb=" P G D 2 " ideal model delta sigma weight residual 1.600 1.597 0.003 2.00e-02 2.50e+03 1.99e-02 However, the issue of broken O3'-P linker persists in Coot when applying real space refine zone on the RNA model after Phenix refinement. So I sent you a separate email with all necessary files that you can have a look at your convenience. Thank you very much! Zixian Li ________________________________ From: Nigel Moriarty Sent: Tuesday, July 7, 2020 4:38 PM To: Zixian Li Cc: [email protected] Subject: Re: [phenixbb] The 5'end guanosine triphosphate is not linked to the rest of RNA model by Phenix refine Zixian Li Some things that immediately require comment. 1. Coot is a separate tool from Phenix and it therefore does not know the things that phenix.refine knows. It does not use the automatic linking or restraints that Phenix uses. You need to supply the restraints that you want Coot to use. Unfortunately, it does not read the params files that Phenix uses. It relies on its internal coding to decide what is linked and what is not. Also http://phenix-online.org/newsletter/CCN_2016_01.pdf#page=10 2. Using the same data_link name "p" is not necessary so I would avoid it. 3. The only surefire way to know if a link has occurred in the refinement is to look in the .geo file. You may have some info in the log file also. Having said that, I'm happy to look into the problem if it persists and you send me the files. Cheers Nigel --- Nigel W. Moriarty Building 33R0349, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : [email protected] Fax : 510-486-5909 Web : CCI.LBL.govhttp://CCI.LBL.gov On Tue, Jul 7, 2020 at 1:09 PM Zixian Li mailto:[email protected]> wrote: Dear Phenix Community, I'm at beginner level of using Phenix. I have difficulty to refine a protein-RNA complex in Phenix. I refined the protein model without RNA first, then I built the RNA model into the difference density map in Coot through Calculate>Other Modelling Tools>Ideal DNA/RNA, where I input the sequence and fit nucleotides into the density. At the 5' end it's guanosine triphosphate (GTP) but the modeler tool in Coot only generates nucleoside monophosphate. So I built the RNA model from the second nucleotide at 5' end downstream until where RNA density was too poor to model. I did the real space refine zone in Coot to fit this main part of the RNA into its density the best possibly. For the GTP at 5' end, I realized there is GTP.cif restraint file in Phenix monomer library. So I obtained an arbitrary pdb file for GTP using eLBOW and corrected atom names matching the definition in GTP.cif file. Then I can also fit GTP into the RNA density at 5' end using real space refine zone in Coot. Now I have a broken RNA model with GTP unlinked to the ribose-phosphate backbone. For Phenix refinement, I supplied a cif_link_GTP-G.params file to define the linkage for the two RNA chains in the complex: refinement.pdb_interpretation { apply_cif_link { data_link = p residue_selection_1 = chain C and resname GTP and resid 1 residue_selection_2 = chain C and resname G and resid 2 } apply_cif_link { data_link = p residue_selection_1 = chain D and resname GTP and resid 1 residue_selection_2 = chain D and resname G and resid 2 } } After refinement, I noticed O3' of GTP appears connected to P of the following nucleotide G in the refined model in Coot. However, if I do the real space refine zone in Coot, the linkage becomes broken, i.e., I cannot refine the RNA model as a whole: Coot only allows separate real space refine for GTP and the rest. It seems Coot still cannot recognize GTP and the rest as one body. In the refinement settings there is a button for Automatic linking options. Interestingly, I noticed if I leave the automatic covalent linking as default, Phenix writes Link records in the output pdb after refinement. But it also links one of the protein residues to the GTP in chain D. In order to avoid the unwanted intermolecular linkage, I clicked Link none for Automatic linking options, then there is no any link records written in the output pdb despite of the defined linkage in cif_link_GTP-G.params file. So it seems for me Phenix ignores the input cif_link_GTP-G.params file, it writes Link records only if the Automatic linking option is activated. I worried whether it could be due to the syntax of the cif_link_GTP-G.params file. So I tried the following syntax in two separate files, the same issue was observed. cif_link_GTP-G_chC.params refinement.pdb_interpretation.apply_cif_link { data_link = p residue_selection_1 = chain C and resname GTP and resid 1 residue_selection_2 = chain C and resname G and resid 2 } cif_link_GTP-G_chD.params refinement.pdb_interpretation.apply_cif_link { data_link = p residue_selection_1 = chain D and resname GTP and resid 1 residue_selection_2 = chain D and resname G and resid 2 } I also need to point out that p linkage is pre-defined in the list of links (mon_lib_list.cif) in Phenix monomer library. I apologize for this long explanation of the problem. I hope it is clear. I really appreciate your advice in advance. Sincerely, Zixian Li _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]mailto:[email protected]