Thanks a lot Roger Rowlett for your very detailed response. I got the SG-Zn refinement to work exactly as you detailed. The key was to rebuild the Cysteines in coot keeping in mind the 2.3 Å coordination distance and not get put off by coot SS bonding things while I tried to get the Cys-SG's in place . So after some fixing in coot -all Zns were 2.0 to 2.5 Å away from a Cys-SG going in to refmac and then your approach worked perfectly.
So I had to: Rebuild and put the Cys-SG within 2.3 A of the Zinc. Remove the SSBOND definitions from the pdb file in an editor Run the mock refinement with refmac to have it detect the SG-ZN coordination Merge the cif file with my ligand cif file. Run the restrained refinement in coot I had just one question. I am imagining that the Cys SG-Zn restraint can be added to the standard refmac monomer dict or I should be able to simply reuse this dummy run cif file everytime I refine a structure with a SG-Zn coordination. Cif files are reside number agnostic is that true. Also since the refmac dict has the ZN defined , how come coot does not have the cif defined? I am assuming I can similarly make coot coordination and Zn ion aware Regardless, thanks again for your detailed response I have the refinement working flawlessly. Hari On Thu, May 13, 2010 at 8:53 PM, Roger Rowlett <[email protected]> wrote: > 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 <[email protected]> > 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 > > >
