Re: [ccp4bb] LINKR vs LINK ? procedure for modeling coordinated Zn+2

[Roger Rowlett]

Hari, Before starting your dummy refmac job to write out the appropriate cif file, you will need to use Coot adjust the positions of the Cys and other Zn-ligand side chains to appropriate positions (consisatent with your electron density maps, of course) to coordinate to Zn. Typical Zn-S bonds are 2.3 A, Zn-O or Zn-N bonds are about 2.0 A. You have to do this in order to break the apparent S-S disulfide bond. You don't have to get the bond distances perfect for refmac to figure it out. You can't do a real-space refinement of the metal ligands in coot because it does not know about the metal ion you have separately added, and will instead try to insert the ligands into the electron density contributed by the zinc. Do the ligand readjustment manually to put everything in to a reasonable place for refmac to start from. Save this as your pdb file with your zinc ion merged in to start the refmac job. Before running refmac, ensure there are no disulfide or other undesired linkage records in your pdb file. Remove them in your favorite text editor as required. I can't remember at the moment if S-S bonds are listed as a LINKR, LINK or as some other type record, but it should be obvious in the PDB file. Just remove the offending S-S bond record. Then start your dummy refmac job, which will fail and generate a cif file with all the metal ligand linkage information. If you have positioned ligands properly, you should see in this cif file definitions for all Zn-ligand linkages and nothing else. Remove any blocks in the cif file that are not linkages you would like to incorporate into your refinement restraints, if necessary. Now run your refmac refinement, using the cif file as your input library with your metal ligand restraints. If the metal ligand distances are reasonable, refmac should pick them up and refine them with proper restraints. Refmac will write the necessary LINKR records into your PDB file for all future refinements of this file. We do this routinely for Zn-metalloenzymes and it works just fine with 2.5-2.9A data. Coot doesn't make a real link between sulfurs: it is just adding a bond for any atoms that are within reasonable bonding distance of each other. If you rotate the sulfur atoms away from each other, Coot will "break" the bond for you. On the other hand, if you leave a linker record in the pdb file for a S-S bond, then refmac will restrain this bond throughout refinement no matter what else is present. Good luck. You should be able to get to to work out properly. Cheers, Roger Rowlett On 5/13/2010 7:31 PM, hari jayaram wrote: Hello Eleanor and Roger Rowlett and everyone, I am still having a tough time with coordinated metal refinements in refmac ( and coot ) I followed Rogers suggestion of creating a pseudo refmac run and then using the output cif definition for Cys -SG Zn as an input for further refinement. Even If I did input this cif , I see that my tetra-coordinated Zinc is pushed aside and my Cysteine SG - are disulphide bonded to each other creating a tetrahedral arrangement. One caveat . The Resolution of the data is no all that spectacular (2.5) so the whole assembly appears rather blobby with three clear Zn spheres like I would expect in the DELFWT maps before I model in the Zn. The other un-related problem is that coot seems to also want to bond Cysteines to each other when I do the real space refinement, but I think thats because SG-Zn restraints are not part of the coot dictionary .So the problem may very well be in the lack of a record in the pdb output by coot that says ..dont disulfide bond these Cys. I did check there were no explicit disulphide links defined or LINK or LINKR. So I am still nor refining these Cys-Zn links and have them mangled in my pdb, so any pointers will be greatly appreciated. Thanks for your help in advance Hari On Thu, May 13, 2010 at 12:50 PM, Eleanor Dodson <c...@ysbl.york.ac.uk> wrote: Has anyone answered this? Eleanor hari jayaram wrote: Hi Am trying to build coordinated Zn+2 by CYS -SG atoms and have it refine properly in refmac5.5 I added ZN ions in Coot at the Fo-Fc peaks. Then I defined manually the coordination relations in a text editor using LINK records. the ions were placed in the same chain that was doing the coordination. LINK        ZN    ZN A1001                 SG  CYS A 487     1555   1555  2.34 I found that some of these ions refined well using refmac 5.5 and the resulting pdb had LINKR records in place indicating the coordinating interactions. For some of the Zn atoms however , the ions did refine , but coot still was covalently disulphide bonding the Cys to each other rather than coordinating the ZN and there were no LINKR records in the refmac refined pdb file. I know these interactions are legitimate because it is a known structure.Should I manually create LINKR records instead of LINK records after modeling in the ions? What is the correct procedure to model coordinating ions and have refmac treat them appropriately. Thanks in advance. Hari