10.1. HEADER records

HEADER records are copied from the PDB input file to lineprinter GRINLOUT for information. There should only be one HEADER in the PDB file, and it should be at the top of the file from where it is copied to the top of the GRINKOUT file. If more than one HEADER is present, the last one is copied to GRINKOUT.

The HEADER may contain upto 60 characters, including spaces and the first six characters which must be 'HEADER'. If a more detailed description of the GRID run is required, comments may be included in the command file (grid.in).

It is suggested that the text of the Header should not begin immedietely after the word HEADER. It may always start after at least 16 empty character positions thus:

HEADER                There are 16 blanks first

This convention is proposed because the first 16 character positions are reserved for a Password in some old copies of Programme GRID. .

10.2. ATOM records

ATOM records for Programme GRIN should comply with the following input format where I is an integer number which identifies this particular record in Programme GRIN:

 100 READ (INKO,120) ATOM, NPDB(I),ATM(I),ALT(I),ACID(I),
    +ISUB(I),NRES(I),INSERT(I),X(I),Y(I),Z(I),OCCUP(I),BVAL(I)
 120 FORMAT (6A1,I5,1X,A4,A1,A3,1X,A1,I4,A1,3X,3F8.3,2F6.2,4X)

This is a standard Protein Data Bank format statement. In fact, however, the actual input format for GRIN is slightly different:

 FORMAT (6A1,I5,A5,A1,A4,A1,I4,A1,3X,3F8.3,2F6.2,4X)

but the difference should not be exploited, and A5 should not normally be the input format for ATM. It should be 1X,A4 as shown in Format statement 120 above.

The names of the variables in ATOM records have the following meanings:

10.3. ATOM records for hydrogens

The original conventions of the Brookhaven Protein Data Bank did not provide for the hydrogen atoms of biological macromolecules, because X-ray studies at that time could not observe those hydrogens. However modern X-ray and NMR methods and molecular dynamics computations, often yield the hydrogen coordinates of proteins. Programme Grid will therefore use this information when it is provided, as described below.

Extra ATOM records must be included in the PDB file for a macro-molecule, in order to define the positions of the hydrogens. It must be emphasised that these are ATOM records as defined by the conventions of the Brookhaven Data Bank. However, there is no particular Brookhaven convention for hydrogen ATOM names, and the Molecular Discovery Programmes will thereford regard any ATOM whose name includes the character 'H' as a potential hydrogen. The Programmes will then test the position of the ATOM in the macro-molecule, in order to establish if it really is a hydrogen atom.

By way of an example one may consider the first ATOM record for an arginine residue in a Brookhaven PDB file for a protein. This record would normally be:

 ATOM     68  N   ARG     5     -11.579   9.499   1.574  1.00  0.00

and it would be followed by ten more ATOM lines for the CA C O CB CG CD NE CZ NH1 and NH2 heavy atoms of arginine. However an additional thirteen ATOM records would be required in the PDB file if the hydrogen coordinates were provided, starting with:

 ATOM     69  HN  ARG     5     -11.634   9.851   2.485  1.00  0.00

for the hydrogen which is bonded to the protein backbone amide nitrogen, and finishing with:

 ATOM     89 HH22 ARG     5     -17.684  12.304   4.077  1.00  0.00

Users should note the following points:

• The hydrogen ATOMS of each residue must be placed in the same part of the PDB file as the corresponding heavy ATOMS. They should come after the first heavy ATOM of their own residue (which is normally its protein backbone nitrogen) and before the last heavy ATOM of the same residue. In particular, a hydrogen should never be the first or the last record in the file as a whole. This is important.

• Within these limits, the sequence of hydrogen ATOMS does not matter.

• The ATOM name of each hydrogen must include the character 'H'. Note that hydrogen names are subject to the normal limitation in Brookhaven PDB files, that only two characters may come after the symbol for the element. Thus a name like HND2A would be unacceptable, because there are three characters after the N.

• Programme GRIN will still detect any extra ATOM records which have been incorrectly added to the PDB file by mistake, even if the ATOM name of the incorrect record contains the character 'H'.

• The Programmes will correctly recognise NH1 and NH2, for example, as the regular Brookhaven names of nitrogen ATOMS in arginine, and will not confuse these ATOM names for hydrogens just because they have names which include the character 'H'.

• Every ATOM name in a residue must be unique. Because one of the side-chain nitrogen ATOMS of arginine is called NH2 according to the conventions of the Protein Data Bank, one must not call the pair of hydrogens which are bonded to this ATOM by the names NH1 and NH2, because these would be duplicate names.

• PDB files may contain explicit coordinates for a potentially tautomeric hydrogen, such as a serine hydroxyl hydrogen for example. In this case Grid will assume that the hydrogen is mobile, and that it can rotate around its C-C-O-H torsion angle. (The 'Type' of the oxygen (OG) ATOM must be changed to Type 4 in the GRINKOUT file if the hydrogen position is to be fixed in its reported position).

• Problems may arise if the PDB file contains some of the hydrogens in a protein as ATOM records, but other hydrogens are not represented. We have tried to cover all such possibilities by corrective procedures or Warning Messages. However, there are so many ways in which hydrogens can be selected for inclusion, that we cannot be certain if all such combinations have been dealt with.

• Please note that the hydrogens of arginine, for example, are still ATOMS in the arginine residues. They must therefore have the same amino-acid name (ARG) and residue number and chain identifier as the heavy atoms of the arginine. Some Users have wrongly used the amino-acid name HYD (instead of ARG) for the hydrogens, or have omitted the residue number, and this could lead to errors. The chain identifier, of course, is only required if the heavy ATOMS of the whole macromolecule have an identifier; it is normally omitted when there is only one subunit in the Target.

10.4. TER records

'TER ' records may just consist of the three capital letters TER at the start of a line. This record shows that the carboxy terminus of a protein chain has been reached. The amino-acid preceding the TER record should therefore contain an extra oxygen (OXT) atom which completes the carboxy terminal group. Provision is made for this OXT atom in datafile GRUB, but it is only called by Programme GRIN if the amino-acid in the PDB file is followed by a TER record.

There should not be a TER record at the start of the PDB file, before the first amino-acid. However, a second protein sequence may follow after a TER record, and the N-terminal nitrogen of this second protein will automatically be treated as a terminal amino group; i.e. it will be a cationic nitrogen, and therefore different from the other backbone amido nitrogens of the protein. Any number of proteins may follow one after the other in this way, but they should not have individual HEADER records.

TER records should not be used at the end of other types of macro-molecular sequence such as DNA. They should not be used after amide -CONH2 termini. They are restricted to the unprotected carboxy terminus of proteins alone.

TER records should not be used after hetero-atoms. Hetero-atoms must appear together at the end of the PDB file, after all the protein ATOMS have been input. If the protein sequence ends in a carboxy group it should be followed by the usual TER record which may then be followed by any HETATMS.

10.5. NAMIDE records

'NAMIDE' records are not part of the standard Protein Data Bank format. However it may sometimes be necessary to force a backbone nitrogen into the amido form. This would be the case if some amino-acid residues were missing at the start of a sequence, because the first amino-acid is normally treated as an N- terminal unless an NAMIDE record is prefixed (see below).

The two consecutive records:

 TER
 NAMIDE

may be used if one protein sequence in the PDB file finishes correctly at the carboxy terminal, but the next sequence has missing residues at its N-terminal end. Please note that there should be no HEADER record at this point.

NAMIDE records should not be used with other types of macromolecular sequence. They are restricted to proteins and peptides alone.

10.6. NTER records

'NTER ' records are not part of the standard PDB format, but may be used in a PDB file in order to force the next following amino-acid into the N-terminal amino form. Thus an NTER record has the opposite effect to NAMIDE (see above). Neither of these should be used in conjunction with hetero-atoms. At the start of the first protein in the file, the first amino-acid is always treated as an N-terminal unless prefixed by NAMIDE.

NTER records should not be used at the end of other types of macro-molecular sequence. They are restricted to the amino terminus of proteins alone.

10.7. HETATM records

'HETATM' records can be used to define the properties of any molecule. Whilst 'ATOM ' records are normally restricted to amino-acids, peptides, proteins, nucleic acids, other biological macromolecules such as sugars and a few other special cases, HETATM records can be used more generally. Drug molecules, water molecules, solvent molecules, ions, inhibitors, cofactors or other ligands may all be defined by HETATM records.

HETATMS and ATOMS can be mixed in the same file, so that water and other ligands can be associated with a chosen macromolecule as a single Target structure. In this case, however, the HETATM RECORDS MUST ALL COME AFTER THE ATOM RECORDS in the PDB file.

HETATMS in a PDB file have essentially the same format as ATOM records, but the value of the first variable is 'HETATM' instead of 'ATOM '. The decision whether to describe a molecule as a collection of ATOMS or HETATMS will be considered in more detail below. ATOMS and HETATMS should never be used together IN THE SAME MOLECULE.

When you run Programme GRIN it will add Energy Variables to the HETATMS in your PDB file, and these Variables will come from a special 'HET' section at the end of Datafile GRUB. This section has Energy Variables for water molecules and other hetero-atom types. The User may add more 'HET' variables to datafile GRUB in order to deal with his or her own interests and applications. This is described in detail below.

It is often necessary to change the names of the HETATMS in the original PDB file, since there is no general agreement on the names to be used for atoms in Hetero-Molecules. There is an editor in Programme GREAT, which may be used in order to do this. It is Item 4 in the first menu of GREAT.

It is not essential to input any ATOMS at all, and the input for Programme GRIN may begin with HETATMS. In this case there should not be a TER record before the HETATMS, but an END record at the end of the file is required.

10.8. Water records

Water molecules associated with the Target in the PDB file may be treated in several different ways. This choice depends on the interests of the User. There is a Table summarising the different treatments at the end of this Section.

You should study the concept of Hydrogen Bonding Type before reading this Section, and should be acquainted with Format Statement 120 above. Note in particular the different and distinctive meanings of the terms ATOM and ATM and ACID:

• ATOM means a whole line in a PDB file; an ATOM record as opposed to a HETATM record.

• ATM is the name of the particular atom in the record; eg ' N ' for nitrogen.

• ACID is the name of the Amino-Acid residue or molecule containing the ATM; eg 'ALA ' for alanine

 H2O....H-N(CH3)3

 H2O....H-N(CH3)3

10.9. END records

'END ' records may just consist of the word END on a line by itself to show that the PDB file has all been input. There should only be one END record, and it should be at the end of the file!

10.10. Inputting a set to Programme GRIN

GRIN normally processes one Target at a time, and this Target may consist of one molecule or a mixture of molecules in PDB format; e.g.: a protein, a bound ligand, some waters and several counter-ions could all be parts of one Target. Alternatively, however, GRIN can be used to process several different Targets as a Set, one after the other in a single GRIN run.

Further information about processing a Set is given above under the headings Set of Targets, and PREPARING A SET OF TARGETS WITH GREAT. More information is given below under INPUTTING A FILE LIST and STUDYING A SET OF TARGETS.