problem of add hydrogens using CHARMM



 Hello:
     I am a newman in CHARMM program, I met a problem when used it to add
 hydrogen to a protein molecule. The input file is the following:
 -------------------------------------
 * Generate PSF and CHARMM coordinate set for
 * the small protein Echistatin
 *
 ! Read in Topology and  Parameter files
 open unit 1 card read name top_all22_prot.inp
 read RTF card unit 1
 close unit 1
 open unit 1 card read name par_all22_prot.inp
 read PARA card unit 1
 close unit 1
 ! Read sequence from the PDB coordinate file
 open unit 1 card read name 1pef.pdb
 read sequ pdb unit 1
 ! now generate the PSF and also the IC table (SETU keyword)
 generate prot setu
 ! read in pdb coordinate file
 open unit 1 card read name 1pef.pdb
 read coor pdb unit 1
 close unit 1
 ! build in hydrogens if using a crystal structure
 hbuild  sele all end
 ! build in missing coordinates using values in
 ! the parameter set
 ic fill
 ic para
 ic build
 ! print all coordinates:  undefined coordinates are indicated as
 ! 9999.00
 print coor
 ! write out the protein structure file (psf) and
 ! the coordinate file in pdb format.
 open write formatted unit 27 name 1pef.psf
 write psf card unit 27
 * PSF for echestatin
 *
 open unit 1 card write name 1pef-allh.pdb
 write coor pdb unit 1
 * echestatin  Structure with hydrogens
 *
 close unit 1
 stop
 --------------------------------------------------------
 And a part of the output file is the following:
 =----------------------------------------------
  CHARMM>    ! Read sequence from the PDB coordinate file
  CHARMM>    open unit 1 card read name 1pef.pdb
  VOPEN> Attempting to open::1pef.pdb::
  OPNLGU> Unit  1 opened for READONLY access to 1pef.pdb
  CHARMM>    read sequ pdb unit 1
  MAINIO> Sequence information being read from unit   1.
  TITLE>  *
           RESIDUE SEQUENCE --    18 RESIDUES
           GLU GLN LEU LEU LYS ALA LEU GLU PHE LEU LEU LYS GLU LEU LEU
 GLU LYS LEU
  ***** Message from SEQRDR ***** THE SYSTEM CONTAINS  7 TITRATABLE
 GROUPS
  THE USER MUST PREDETERMINE THE PROTONATION STATE THROUGH THE SEQUENCE
 AND RTF
  HIS -  0  ASP -  0  GLU -  4  LYS -  3  TYR -  0
  CHARMM>
  CHARMM>    ! now generate the PSF and also the IC table (SETU keyword)
  CHARMM>
  CHARMM>    generate prot setu
  THE PATCH 'NTER  ' WILL BE USED FOR THE FIRST RESIDUE
  THE PATCH 'CTER  ' WILL BE USED FOR THE LAST  RESIDUE
  GENPSF> Segment   1 has been generated. Its identifier is PROT.
  PSFSUM> PSF modified: NONBOND lists and IMAGE atoms cleared.
  PSFSUM> Summary of the structure file counters :
          Number of segments      =        1   Number of residues
 =       18
          Number of atoms         =      328   Number of groups
 =      100
          Number of bonds         =      328   Number of angles
 =      609
          Number of dihedrals     =      882   Number of impropers
 =       43
          Number of HB acceptors  =       28   Number of HB donors
 =       31
          Number of NB exclusions =        0   Total charge =   -1.00000
  CHARMM>
  CHARMM>    ! read in pdb coordinate file
  CHARMM>    open unit 1 card read name 1pef.pdb
  OPNLGU> Unit already open. The old file will be closed first.
  VCLOSE: Closing unit    1 with status "KEEP"
  VOPEN> Attempting to open::1pef.pdb::
  OPNLGU> Unit  1 opened for READONLY access to 1pef.pdb
  CHARMM>    read coor pdb unit 1
           SPATIAL COORDINATES BEING READ FROM UNIT  1
  TITLE>  *
  ** WARNING ** For atom in coordinate file, could not find residue in
 PSF, and is thus ignored:
   SEGID=     RESID=1    RESNAME= GLU  TYPE= N
  *** LEVEL  1 WARNING *** BOMLEV IS    0
  ** WARNING ** For atom in coordinate file, could not find residue in
 PSF, and is thus ignored:
   SEGID=     RESID=1    RESNAME= GLU  TYPE= CA
  *** LEVEL  1 WARNING *** BOMLEV IS    0
  ** WARNING ** For atom in coordinate file, could not find residue in
 PSF, and is thus ignored:
   SEGID=     RESID=1    RESNAME= GLU  TYPE= C
  *** LEVEL  1 WARNING *** BOMLEV IS    0
  ** WARNING ** For atom in coordinate file, could not find residue in
 PSF, and is thus ignored:
   SEGID=     RESID=1    RESNAME= GLU  TYPE= O
  *** LEVEL  1 WARNING *** BOMLEV IS    0
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     1     1 GLU  N
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     2     1 GLU  HT1
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     3     1 GLU  HT2
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     4     1 GLU  HT3
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     5     1 GLU  CA
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     6     1 GLU  HA
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     7     1 GLU  CB
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     8     1 GLU  HB1
  ** WARNING ** After reading, there are no coordinates for selected
 atom:     9     1 GLU  HB2
  ** WARNING ** After reading, there are no coordinates for selected
 atom:    10     1 GLU  CG
  ** A total of   328 selected atoms have no coordinates
  ** MESSAGE **   153 atoms in coordinate file were outside the specified
 sequence range.
  *** LEVEL  2 WARNING *** BOMLEV IS    0
  CHARMM>    close unit 1
  VCLOSE: Closing unit    1 with status "KEEP"
  CHARMM>
  CHARMM>    ! build in hydrogens if using a crystal structure
  CHARMM>    hbuild sele all end
  SELRPN>    328 atoms have been selected out of    328
  PRNHBD: CUToff Hydrogen Bond  distance =    0.5000   Angle =   90.0000
          CuT switching ON HB dist. =     3.5000  OFf HB dist. =
 4.0000
          CuT switching ON Hb Angle =    50.0000  OFf Hb Angle =
 70.0000
          ACCEptor antecedents included
          All hydrogen bonds for each hydrogen will be found
          Hydrogen bonds between excluded atoms will be kept
  NONBOND OPTION FLAGS:
      ELEC     VDW      ATOMs    CDIElec  SHIFt    VATOm    VSWItch
      BYGRoup  NOEXtnd  NOEWald
  CUTNB  = 14.000 CTEXNB =999.000 CTONNB = 10.000 CTOFNB = 12.000
  WMIN   =  1.500 WRNMXD =  0.500 E14FAC =  1.000 EPS    =  1.000
  NBXMOD =      5
  There are        0 atom  pairs and        0 atom  exclusions.
  There are        0 group pairs and        0 group exclusions.
  <MAKINB> with mode   5 found    937 exclusions and    879
 interactions(1-4)
  <MAKGRP> found    291 group exclusions.
  CUToff Hydrogen Bond  distance =    0.5000           Angle =   90.0000
  CuT switching ON HB dist. =     3.5000 OFf HB dist. =    4.0000
  CuT switching ON Hb Angle =    50.0000 OFf Hb Angle =   70.0000
  ACCEptor antecedents included
  dihedral PHI STePs for spin =   10.0000
  cutoff for water acceptor search CUTWat=    7.5000
  WARNING FROM HBUILD:
  Attempt to construct a hydrogen bonded to the atom PROT 1    GLU  N
 with unknown coordinates.
  *** LEVEL  0 WARNING *** BOMLEV IS    0
  BOMLEV HAS BEEN SATISFIED. TERMINATING.
 =====================================================================================================
 I wish you can help me solve this problem. Thanks.
 Regards,
 mao xiang
   ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 |  Xiang Mao                                                     |
 |  Lab of Molecular Regulation for Microbial Secondary Metabolism |
 |  Shanghai institute of Plant Physiology, Academia Sinica        |
 |  300 Fenglin Road, Shanghai, China, 200032                      |
 |  Tel: +86-21-64042090-4791                                      |
 |  Fax: +86-21-64042385                                           |
   ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~