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Re: [ccp4bb] LINKR vs LINK ? procedure for modeling coordinated Zn+2
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Subject: refines Query for non covalent interactions
From: gauri misra kamgauri {- at -} GMAIL {- dot -} COM
Date: 2010-05-14		Next message:
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From: intekhab alam faisaldbg {- at -} GMAIL {- dot -} COM
Date: 2010-05-14


Subject: Re: LINKR vs LINK ? procedure for modeling coordinated Zn+2
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2010-05-14




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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 face="Verdana">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:
cite="mid:AANLkTikZdtreHrU5ZP1MwcDr46P699PLiaZZYjJiQ-uK@mail.gmail.com"
type="cite">
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 <ccp4@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








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