Working With Metal Ions

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One-third of all proteins are "metalloproteins", chemical combinations of protein atoms (carbon, nitrogen, oxygen, hydrogen, sulfur) with ions of metals such as iron, calcium, copper, and zinc. The hemoglobin, for example, that carries oxygen in the bloodstream, is an iron-containing metalloprotein. The metal ions in metalloproteins are critical to the protein's function, structure, or stability. In fact, numerous essential biological functions require metal ions, and most of these metal ion functions involve metalloproteins.

To identify if a protein contains metal ion, bioinfomatic methods such as "zinc finder", and experimental methods such as NMR, SDS/native page can be used.

Running CYANA/AUTOSTRUCTURE for Proteins with Metal Ions

CYANA/AUTOSTRUCTURE can handle structure calculation for protein with metal ions. One can use metal ion attached amino acid or attach the metal ion to protein sequence by using a pseudo long link during CYANA calculations

  1. Check CYANA.lib for special residues: (ION, PL, LL5, LP, CYSZ,HISZ,...,CYS-, CYSS,...).
    1. ION:ion residue contain only one metal ion "ME"
    2. PL/LP: pseudo link residues  from protein to "ION" / "ION" to protein
    3. LL5: psdudo link residues
    4. CYSZ: Zinc attached cystine with cystine.SG convanlently  binds to zinc.
    5. CYSS: cystine residue without "HG" atom
    6. CYS-:  negative charged cystine residue without "HG" atom
  2. Modify the protein's sequence file to have the required special residues included
    1. Using metal ion attached residues (need pay attention to the charge), eg:
 VAL  76
 GLN  77
 CYSZ  78
 PHE  79
 ALA  80
 CYS-  81
 GLY 82 
    1. Using psedo link, eg:
 VAL  76
 GLN  77
 CYSS  78
 PHE  79
 ALA  80
 CYSS  81
 GLY  82

  1. Upl/lol/aco constraints. Use reliable metal-ligand geometry parameters to derive upper/lower distance limits and angle constraints, and treated as addtional manual constraints during CYANA calculation. eg:
 78   CYSZ   ZN      81  CYS-    SG         2.30  5E01
 78   CYSZ   ZN      98  HIS     NE2        2.40  5E01
 78   CYSZ   ZN      105 CYS-    SG         2.30  5E01
 78   CYSZ   SG      81  CYS-    SG         3.65  5E01
 78   CYSZ   SG      98  HIS     NE2        3.75  5E01
 78   CYSZ   SG      105 CYS-    SG         3.65  5E01
 81   CYS-   SG      98  HIS     NE2        3.75  5E01
 81   CYS-   SG      105 CYS-    SG         3.65  5E01
 98   HIS    NE2     105 CYS-    SG         3.75  5E01

Running CNS for Proteins with Metal Ions

The CNS refinement for proteins with metal ions can also be performed with the new WaterrefCNS script, using the appropriate metal ion nomenclature in both the pdb coordinates and distance constraints.

For manual CNS refinement please do the refinement as following:

1.  Set environment for CNS1.1 by run: 

alias cns1 /farm/software/cns/cns_solve_1.1/intel-i686-linux_g77/bin/cns

setenv CNS_TOPPAR /farm/data/gliu/cns1/ 

In addition to the topology and parameter files, the metal ion parameter file is required. An example can be found in "cns_cuttha_cis" with all required input files.

2.  Prepared required files as described above (final.tbl, final cns format PDB files and put in xplorPDB dir with name as sa_#.pdb) except the PDB file should include the metal ions with format according to CNS library cp sa_1.pdb as  template.pdb, input files for creating mtf file Note that alignment is important. eg:

ATOM   1249  OT2 ALA    83      69.296  13.232   5.744  1.00  0.00
ATOM   1250 ZN+2 ZN2   150      63.086  13.789 -10.407  1.00  0.00      zinc

3.  Run generate_h2o.inp once to create temp_h2o.pdb and temp_h2o.mtf. The extra proton atom in the ligand residues, eg. HIS HD1 or CYS S, are removed by editing the generate_h2o.inp; cis proline is also defined here in the generate_h2o.inp (resid is the residue number prior the proline).

{* any special prosthetic group patches can be applied here *}
delete select (name hg and resname cys and resid 61) end
delete select (name hg and resname cys and resid 85) end
delete select (name hd1 and resname his and resid 46) end
delete select (name he2 and resname his and resid 83) end
 patch cisp
           reference=1=( resid 13 )

4.  Edit generate_1.inp to remove the extra proton as did above.

5.  Run, this will run generate_#.inp 20 times, updating each pdb number and this creates cnsPDB/sa_cns_#.pdb

6.  Edit and run re_h2o_cu.inp, the refined pdb is kept in refinedPDB, or

7.  Use PBS to submit cns refinement parallal: eg, type "sh subcns". Before run subcns , make a folder " com" contains the following file. Type getpdb to get refined pdb files in refinedPDB after it finished.

  • PBS submission
  • cutc_h2o.mtf: mtf file created as descrive above
  • topology and parameter files:,,
  • re_h2o_cu.inp: input file for cns refinement