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Chapter 22. Messages from GRIN at run time

Run time messages from Programme GRIN are printed to the lineprinter output file GRINLOUT. They are identified by a number which is prefixed by the Letter N and followed by a suffix. The number is the label of the format statement which prints the message. (Note that the same format statement with the same label may occur in more than one subroutine.) The letter N identifies messages from Programme GRIN, and the suffix indicates the severity of the message or warning.

Messages are self explanatory. They are listed below with further information when need be. However, the following list of messages should NOT be studied by the User until it is necessary to elucidate a particular message.

22.1. Severity of messages and warnings

Messages are divided into three different categories:

Information messages: suffix -I follows the message number.
Warnings:: suffix -W follows the message number.
Errors:: suffix -E follows the message number.

Information messages are intended to draw the User's attention to unexpected features of the input data, the computation or the results. In general, no User action is required.

Warnings from GRIN should be carefully considered by the User. They are not normally fatal, but the associated output file GRINKOUT should not be used as input for the next Programme GRID if there are significant warnings in GRINLOUT. On the contrary, appropriate changes should be made to the input files PDB , and Programme GRIN should be rerun to give a GRINKOUT output file with no significant warnings in the associated GRINLOUT file. It is sometimes necessary to repeat this cycle several times before an acceptable GRINKOUT file is obtained. It may be helpful to change the value of directive LEVL (see below).

Errors from Programme GRIN may not always be fatal, but the associated GRINKOUT file should NEVER be used as input for the next Programme GRID. Appropriate changes should be made to the input, and the above cycle repeated until an acceptable output is obtained.

The User may decide if more or if fewer messages and warnings should be issued. The number of messages is controlled by changing the value of input directive LEVL. When the value of this is low (eg LEVL = 1) many messages will be suppressed. More will be printed to the lineprinter output file GRINLOUT if a bigger value of LEVL is used (e.g. 3 or 4).

A single mistake in the PDB file can give rise to several associated messages, and it may not be easy to interpret them all. However, if some of the errors in PDB are corrected, the others may be cured as well.

Programme GRIN will take significantly longer to run, if there are many mistakes in the PDB imput file. This is because the mistakes can interact with each other, and the Programme may have to interpret the file in several different ways, before deciding that there is a definite mistake. It is better to use a fault-free file in the first place!

When a GRIN run has been completed, there may or may not be a final General Warning at the end of the lineprinter output file GRINLOUT. There will be no General Warning if the run was successful, and in this case the User may decide not to study the individual messages one by one. Otherwise the severity of the final General Warning will give an overall indication of the number of messages in the PDB file, and of their importance.

The run time messages and warnings written to the lineprinter file GRINLOUT are listed over the page.

Note: The following list need NOT be studied by the User, until it is necessary to elucidate a message or warning.

22.2. List of messages and warnings from programme GRIN at run time

22.2.1. N080-W

  (N080-W)  COMPUTATION OF AM1 ATOMIC CHARGES 
            FAILED. THE PROCEDURE WAS REVERTED 
	    TO SAUNDERSON'S ALGORITHM.

The procedure was reverted to the Saunderson's algorithm because the semiempirical method failed. Details are reported in message N090-W.

22.2.2. N090-W

  (N090-W)  OUT OF MEMORY ERROR IN AM1CHRG, or
            INVALID ATOM ERROR IN AM1CHRG, or
            OPEN SHELL ERROR IN AM1CHRG, or
            SCF WAS NOT ACHIEVED ERROR IN AM1CHRG or
            INPUT TOO LARGE FOR AM1 CALCULATION.

A problem occurred when charges were calculated with a semiempirical method. The procedure was reverted to the Saunderson's algorithm.

22.2.3. N100-I

  (N100-I)  PLEASE CHECK DIRECTIVE INAT = ....
            A POSITIVE INTEGER BELOW 100 WAS EXPECTED

INAT is the number of an input channel providing data to Programme GRIN. The Programme will reject channel numbers outside the Range 1-99. If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.4. N110-I

  (N110-I)  THE FOLLOWING VALUES WILL BE USED BY DEFAULT FOR          
            HYDROGEN ATOMS WHICH CANNOT MAKE HYDROGEN BONDS:          
                VDWR=1.15    NEFF=1    ALPH=0.1      Q=0.0            
            YOU MAY SPECIFY ALTERNATIVE VALUES BY DEFINING A          
            HETATM ' H  ' IN THE HET SECTION OF DATAFILE GRUB

Datafile GRUB is normally supplied with a hydrogen ' H ' in the HET section at the end of the Datafile. However, message N110 indicates that this hydrogen is missing from the copy of GRUB which you are using, and so default values have been supplied by Programme GRIN. Perhaps the original hydrogen HETATM has been edited out of the Datafile, and should now be reinstated.

Do not mistake these variables for VDHY, EFHY, ALHY and QQHY which are the values that will be given to hydrogen-bonding hydrogens. The present message N110 relates to hydrogens which do not form hydrogen bonds. We recommend that you reinstate the hydrogen line in Datafile GRUB.

22.2.5. N120-E

  (N120-E)  CHECK PHOSPHATE GROUP AND counter-ion                     
            IN NUCLEIC ACID RESIDUE .....

This error occurs while Programme GRIN is trying to add counter-ions to the phosphate groups of DNA or RNA. There may be an anomoly in the three-dimensional structure of the phosphate or its neighbours, or some atoms may be missing. As a result the counter-ion may not have been inserted correctly in the GRINKOUT output file.

22.2.6. N130-I

  (N130-I)  '!' IS A RESERVED SYMBOL FOR VARIABLE 'ALT'.              
            IT SHOULD ONLY BE USED WITH SYMMETRICAL TARGETS.          
            SEE USER MANUAL UNDER 'EXCLAMATION MARK'.                 
            'KWIK' MUST ALSO BE SET EQUAL TO 1 IN GRID.

The two halves of a symmetrical Target with a diad axis will generate identical GRID maps, and only one half of such a map is actually needed. With higher orders of symmetry it is possible to save even more computing time, by only computing the GRID map for one out of many identical regions.

The exclamation mark may be used to define those regions of the Target which should be omitted from a symmetrical map. The procedure is described in this User Manual under the definition of the ALT variable, and the directive KWIK.

When dealing with symmetrical Targets in this way, it is necessary to set the value of KWIK equal to 1 (or 2) during the subsequent GRID runs, and the last line of this information message is printed as a reminder of this requirement.

The acceptable values of KWIK are restricted in some situations. It must not have the value 1 or 2 when a multi-atom Probe is being used. See the main manual entry under Directive KWIK for full information.

22.2.7. N140-W

  (N140-W)  INSUFFICIENT POINTERS TO ATOM OR HETATM .....

This error occurs while Programme GRIN is trying to compute the position of one of the hydrogen atoms. It may be due to unacceptable Target geometry, such as two atoms in identical positions, or the absence of a neighbouring atom which is needed as a pointer.

Error N140 is not fatal, but the User should make a careful note of the atom details and should try to correct the PDB file. If this cannot be done, he should exercise caution when studying results in nearby regions of the Target.

The same eror number N140 may be generated by several different subroutines, each of which deals with one type of hydrogen geometry in the Target.

22.2.8. N150-I

  (N150-I)  THERE IS A POTENTIAL GLYCOSYLATION SEQUENCE               
            AT RESIDUES:   ...   ...   ...

Proteins may be glycosylated at places where the sequence is: ASN-X-SER or ASN-X-THR and one of these sequences has now been detected in the Target. See also messages N770 and N1050 below.

22.2.9. N160-W

  (N160-W)  THE ATOM NAME IN THIS WATER RECORD ......                 
            HAS BEEN CHANGED TO .....                                 
            PLEASE CHECK THIS IN THE OUTPUT

Water molecules in the PDB file may be treated as ATOM or HETATM records:

When water is an ATOM the name of the atom must be ' O ' as specified by the Brookhaven PDB format.
When water is a HETATM the name of the atom may be ' OH2' or ' OHH' or ' O2 '. The choice between these alternative atom names for a HETATM water, will influence the way in which the water molecule is treated. ' OH2' is the default name for a HETATM water.

It is important to treat waters consistantly. ATOM waters (' O ') and HETATM waters (' OH2') receive the same standard Energy Parameters. However HETATM waters ' OHH' and ' O2 ' get special treatment. See the list of Contents under the heading 'WATER RECORDS' for details.

This message is printed because there is an inconsistancy in the water record. For instance you may have given the atom name ' O ' to a HETATM record, when it should have been used for an ATOM water. Or again, you may have called a HETATM water ' OHH' without supplying the two associated hydrogen records which are an obligate requirement that is needed to complete the whole ' OHH' water molecule.

Note on atom and molecule names: An ATOM (or HETATM) record contains both an atom and a molecule name. These are ATM(I) and ACID(I) in Format Statement 120 above (see under Format 120). They should not be confused with each other. This message N160 refers to atom (ATM(I)) names.

22.2.10. N170-W

   (N170-W)  PLEASE CHECK ALL THE WATER RECORDS

This warning is printed because there are many inconsistancies in the water records in the PDB file. You will already have had several more detailed messages. We suggest you comprehensively revise the water nomenclature in your input file, in order to be sure that waters are treated the way you want.

If you have been running Programme GRIN with a low value of Directive LEVL (eg 1 or 2) you can get more information by resetting LEVL to 3 or 4 and rerunning the Programme.

22.2.11. N180-W

  (N180-W)  THE MOLECULE NAME OF THIS WATER RECORD: ........          
            HAS BEEN CHANGED TO THE STANDARD BROOKHAVEN NAME: 'HOH '

The recommended Brookhaven name for a water molecule is 'HOH'. The name of this water has been changed accordingly. Message N180 is only sent for information, and you probably do not need to check the altered record.

Note on atom and molecule names: An ATOM (or HETATM) record contains both an atom and a molecule name. These are ATM(I) and ACID(I) in Format Statement 120 above (see under Format 120). They should not be confused with each other. This message N180 refers to molecule (ACID(I)) names.

22.2.12. N190-W

  (N190-W)  PLEASE CHECK THIS RECORD: .................

This warning is printed because there appears to be an inconsistancy in the record. Perhaps the molecule name implies a water molecule, but the atom name implies something else.

When water is an ATOM record, the name of the atom must be ' O ' as specified by the Brookhaven PDB format. The name of the molecule may be 'H2O' or 'HOH' or 'OH2' or 'WAT'.

When water is a HETATM the atom name may be ' OH2' or ' OHH' or ' O2 '. The choice of atom name for a HETATM water determines the way in which that water will be treated by Programme Grid. However, the molecule name for a HETATM water must be: 'HOH'

Also see message N830 below.

22.2.13. N200-I

  (N200-I)  PLEASE CHECK DIRECTIVE INKO = ....                        
            A POSITIVE INTEGER BELOW 100 WAS EXPECTED

INKO is the number of an input channel providing data to Programme GRIN. The Programme will reject channel numbers outside the Range 1-99. If you wish to alter these limits please contact us at http://www.moldiscovery.com .

22.2.14. N210-E

  (N210-E)  ERROR WHEN CHANGING DEFAULT DIRECTIVES                    
            FOR PROGRAMME GRIN.   The acceptable                      
            directives for Grin are:                                  
               IDIR   INAT   INKO   LOUT   KOUT                       
               ALHY   IHVA   LEVL   MOVE   NEHY                       
               QQHY   VDHY   IEND                                      
            PLEASE CHECK YOUR INPUT.

This message usually indicates that one of the directive names have been mistyped. Therefore start by checking the spelling in the command file for GRIN.

The use of directives in the command file is not obligatory because default values are given. If directives are used they should be input through channel IDIR. This channel has a default value IDIR = 5 when Programme GRIN is supplied. IDIR itself can be changed by editing the Programme, and recompiling, but it cannot be changed by a directive at run time.

All the other directives may be changed at run time. Each of the directives to be changed should appear as part of the input, on a line by itself, followed by the new value of the directive. This value will be a number. Directives which begin with the letter I, J, K, L, M or N have integer values.

All the directives are described in detail above. Integer directives ( IDIR, IHVA, INKO, INAT, LOUT, KOUT and LEVL) should have integer values, and real directives ( ALHY, EFHY, QQHY and VDHY) should have real values with a decimal point. The list of directives should always finish with IEND on a line by itself. IEND must always be used, even if no other directives are input. IEND does not have any associated numerical value. Error N210 is fatal.

Note: The Directive IHAC has been implemented only in the Programme GREATER. All Users who are more practice using GREAT may manually add a line in the command file GRIN.IN.

22.2.15. N220-W

  (N220-W)  PROBLEM DEALING WITH THE HYDROGENS                        
            WHICH ARE BONDED TO ATOM .....

In this case Programme GRIN is not able to compute the coordinates of a hydrogen atom which is bonded to an ATOM or HETATM of the Target. This error can occur if one of the backbone nitrogens of a protein has not been given the name ' N ', but has some other incorrect name. It may also occur if the connectivity matrix in GRINKOUT has been corrupted, or if a new hydrogen Type number has been introduced into GRUB without making appropriate adjustments to the Programmes.

This warning may occur if there is a break in a protein sequence as given in the PDB file. Consider for example this sequence where the backbone nitrogens are named from a to h:

Now, if part of the sequence is missing:

there is insufficient information from which to make an unequivocal assignment of the position of the hydrogen bonded to Nf. Two bond vectors from two carbons to this nitrogen are required, but only one such vector can be defined after the break. Message N220 is therefore issued.

This message may sometimes be ignored, unless you are interested in the local structure near the break in the protein chain. The important thing is to ensure that the nitrogen is NOT treated as a cationic N-terminal atom, because the introduction of a spurious positive charge could influence the interactions of a charged Probe in a misleading way. See NAMIDE in the manual.

22.2.16. N230-E

  (N230-E)  ERROR DURING INPUT OF ACID:.....FROM DATAFILE GRUB

This error occurs during the input of datafile GRUB, if Programme GRIN cannot interpret the first line of the entry for an amino-acid type, i.e. the line with the name of the acid and the number of atoms. It often occurs at the first GRUB record if that datafile has been incorrectly assigned. This error is fatal.

22.2.17. N240-I

  (N240-I)  ATOM ... IN MOLECULE ... WILL BE TREATED AS THE           
            5* TERMINAL OXYGEN.

This information message is issued when the Target is a DNA or RNA molecule, and Programme GRIN is processing atoms at the 5* terminal. In this special situation the 5* terminal phosphate group is only bonded to one ribose ring, and the properties of this oxygen atom must therefore be adjusted. Message N240 is printed for information.

22.2.18. N250-I

  (N250-I)  ATOM ... IN MOLECULE ... WILL BE TREATED AS THE           
            3* TERMINAL HYDROXYL GROUP.

This information message occurs when the Target is a DNA or RNA molecule, and Programme GRIN is processing atoms at the 3* terminal. The 3-substituent of the ribose is therefore a free hydroxyl group, and its Energy Variables and those of the neighbouring 3-carbon atom must be adjusted. Message N250 is printed for information.

22.2.19. N260-W

  (N260-W)  HETATM ...... IN MOLECULE .........                       
            IS TYPE 96.   THIS TYPE NUMBER IS RESERVED FOR THE        
            OXYGEN OF WATER, WHEN THE HYDROGEN POSITIONS HAVE ALSO    
            BEEN SUPPLIED.   PLEASE CHECK YOUR DATAFILE GRUB.

Type 96 is uniquely associated with the HETATM name 'OHH' which defines the oxygen atom of a water molecule in the Target, when the User has also supplied the coordinates of the two hydrogens bound to that oxygen. This warning is issued if a User has edited Datafile GRUB, and has mistakenly associated the Type number 96 with some other HETATM name.

22.2.20. N270-E

  (N270-E)  PARAMETER MAXHMO IS ONLY DIMENSIONED UPTO ...             
            HETERO-MOLECULES AND THIS NUMBER HAS NOW BEEN             
            REACHED OR EXCEEDED.

Programme GRIN calculates the charges on the HETATMS of one hetero-molecule at a time, and then continues with the next hetero-molecule. 24000 is the maximum number of hetero-molecules which it can deal with, and this limit has now been exceeded; please contact us at http://www.moldiscovery.com .

22.2.21. N280-W

  (N280-W)  PLEASE CHECK THE HYDROGENS WHICH ARE BONDED TO            
            HETATM ......     THEY WILL NOT HAVE CORRECT              
            COORDINATES, UNLESS YOU SPECIFIED THEIR POSITIONS         
            IN YOUR PDB INPUT FILE.

Your PDB file contains water molecules with Type 96 oxygen HETATMS. These are oxygens for which the positions of the two bonded hydrogen atoms of the water must be supplied in the PDB file. The present warning is to remind you of this requirement, so that you can provide the necessary HETATM records for those hydrogens.

If you do not want to specify the hydrogen records, then Type 96 oxygen should not be used. Try a Type 95 oxygen (which has the HETATM name OH2) instead. Note this is the atom name for the oxygen; it is not the molecule name which appears as a different field of the HETATM record.

This warning will only be given twice. The third warning will be briefer, and the warnings will be discontinued if there are more Type 96 oxygens without hydrogens in your PDB file. You have been warned.

Also see Warning N590 below.

22.2.22. N290-W

  (N290-W)  PLEASE CHECK THE CHARGES IN HETERO-MOLECULE ...           
            IT CONTAINS AN UNRECOGNISED ATOM OF UNKNOWN               
            ELECTRONEGATIVITY.

Programme GRIN calculates the electrostatic charge distribution in hetero-molecules from the electronegativities of the individual hetero-atoms. It has a list of electronegativity values for various elements and a element has now been encountered which does not occur on this list.

You should start by checking the names of all the HETATMS in this hetero-molecule. In particular:

Each name should include the correct symbol for the element; eg S for Sulphur or SN for tin.
The symbol should be in CAPITALS; eg SN but not Sn.
There should not be any character in the column in front of the symbol; eg S for Sulphur but not AS, although one or two characters (but not more) are permitted immediately after the symbol.
Finally, the symbols for the elements must be right-justified, so that Sulphur, Tin and Arsenic might be listed like this in the file:
SA SN1 AS10
meaning Sulphur-A Tin-1 and Arsenic-10

As a default procedure, the unknown hetero-atom will have been given the electronegativity of a hydrogen atom, during this run of Programme Grin. However, the HETATM name should be corrected in the PDB file, and Grin should then be run again.

22.2.23. N300-I

  (N300-I)  PLEASE CHECK DIRECTIVE LOUT = ....                        
            A POSITIVE INTEGER BELOW 100 WAS EXPECTED

LOUT is the number of an output channel from Programme GRIN. The Programme will reject channel numbers outside the Range 1- 99. If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.24. N310-E

  (N310-E)  ERROR WHILE READING DATAFILE GRUB.                        
                    ....IS ONE TOO MANY MOLECULES

Programme GRIN is dimensioned to deal with upto 200 different Recognised Molecule types in file GRUB, and this limit has been exceeded.

This error is fatal. If you want to have more Recognised Molecules in GRUB please contact us at http://www.moldiscovery.com .

22.2.25. N320-W

  (N320-W)  ATOM ... IN MOLECULE ... WILL ONLY BE                     
            ASSIGNED VAN DER WAALS PROPERTIES.

Programme GRIN has not found the expected number of covalently bonded neighbours to a hydrogen-bonding atom. For example, a phenolic oxygen should have one bonded neighbour, or two including the hydrogen, and GRIN may have found three.

When this happens, GRIN may not be able to compute the positions of any hydrogen-bonding hydrogen atom, and may not be able to determine the hydrogen bonding properties of the atom. In these circumstances, the atom will be treated as if it does not make any hydrogen bonds at all, and this warning N320 is printed.

The User should thoroughly check the local geometry round this atom, and the atom and molecule names in the PDB file. He or she should then rerun GRIN, and make sure that the atom has been given the correct hydrogen-bonding Type. The electrostatic charge of the atom and any bonded hydrogens should also be checked.

22.2.26. N330-W

  (N330-W)  PLEASE CHECK 3* TERMINAL STRUCTURE IN MOLECULE ...

There is an unexpected feature of the molecular structure. Perhaps some atoms are missing, or have the wrong coordinates, or have not been assigned acceptable names, or have been assigned the wrong molecule or residue name or number.

The User should search for any such unacceptable features in the PDB file, and should then rerun GRIN. If this warning is still issued he or she should assess the atom charges before proceding with Programme GRID.

22.2.27. N340-W

  (N340-W)  ATOM ... IN MOLECULE ... WILL BE                          
            TREATED AS A CARBONYL OXYGEN.

Programme GRIN has been determining the environment of an oxygen atom. An ether oxygen (Type 28) was expected, but this oxygen has only one neighbouring atom within covalent bonding distance. In these circumstances the oxygen will be treated as a carbonyl oxygen. The User should study the environment of this particular oxygen, in order to find out why message N340 occurred. The electrostatic charge distribution should also be checked.

22.2.28. N350-I

  (N350-I)  counter-ion ADDED TO NUCLEIC ACID RESIDUE .....

This message confirms that Programme GRIN has successfully added a counter-ion to a phosphate group of DNA or RNA.

22.2.29. N360-W

  (N360-W)  A SMALLER VAN DER WAALS RADIUS WAS EXPECTED FOR.          
            ATOM ........  PLEASE CHECK

The name of this atom includes the letter H in a manner which suggests that it may be a Hydrogen atom. However, its van der Waals radius is too big for Hydrogen, and this warning is therefore printed.

22.2.30. N370-E

  (N370-E)  HETERO-ATOM .... HAS NOT BEEN GIVEN A MOLECULE NAME.      
            IT WILL BE TEMPORARILY ASSIGNED TO MOLECULE 'JUNK'.       
            IT MUST BE GIVEN A PROPER MOLECULE NAME, BECAUSE          
            HETATM CHARGES CANNOT BE CALCULATED AT PRESENT.

Programme GRIN calculates the charges on the HETATMS of one hetero-molecule at a time, and then continues with the next hetero-molecule. Every HETATM must therefore be asigned to the correct hetero-molecule. This assignment is made on the basis of the hetero-molecule name and number and the insertion character, as defined by variables ACID and NRES and INSERT of the HETATM. For instance, if your file contains one morphine cation; one chloride anion and three water molecules they might be named like this in your PDB input file:

   MOR 1
   CHL 1
   WAT 1
   WAT 2
   WAT 3

However, acceptable values of ACID and NRES and INSERT have not been specified for this HETATM, and so Programme GRIN does not know the hetero-molecule to which the HETATM should be assigned. In some cases it might be able to deduce the correct molecule by determining the connectivity between the atoms, but this can be an unreliable method if the Target has been flooded with water molecules. It is therefore necessary for the User to edit the PDB file, and give ACID and NRES (and/or INSERT) values to this and every HETATM. Programme GRIN should then be rerun on the corrected PDB file.

22.2.31. N380-I

  (N380-I)  EXTRA ATOMS FOUND WITHIN COVALENT BONDING RANGE           
            NEAR ATOM  ...     NEAREST ATOMS CHOSEN.

When Programme GRIN computes the positions of the hydrogen atoms which are bonded to a heavy atom of the Target molecule, it starts by identifying all the other atoms which are bonded to that heavy atom. It will need to know the positions of some of these nearby atoms in order to calculate the hydrogen positions, but it sometimes identifies more neighbours than it actually needs. This message will then be printed for information, if the value of Directive LEVL > 4

A high value of Directive LEVL is required to force this message, because the finding of extra atoms does not constitute an error. However, this message may be useful for checking purposes.

22.2.32. N390-W

  (N390-W)  A BIGGER VAN DER WAALS RADIUS WAS EXPECTED FOR.           
            ATOM ........  PLEASE CHECK

The name of this atom does not include the letter H in an appropriate manner for a Hydrogen atom. However, the van der Waals radius would be appropriate for Hydrogen, and this warning has therefore been printed.

22.2.33. N400-I

  (N400-I)  PLEASE CHECK DIRECTIVE KOUT = ....                        
            A POSITIVE INTEGER BELOW 100 WAS EXPECTED

KOUT is the number of an output channel from Programme GRIN. The Programme will reject channel numbers outside the Range 1-99. If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.34. N410-E

  (N410-E)  STILL MORE VALUES SELECTED, ASSUMED, OR GUESSED

Sometimes there are no suitable Energy Variables in datafile GRUB for the atoms in file PDB. An attempt may then be made by Programme GRIN to select or assume or guess suitable parameter values. Warnings are only printed on the first twenty-five occasions when this occurs, after which this warning N410 (or warning N660) is printed once.

22.2.35. N420-E

  (N420-E)  ERROR DURING THE INPUT OF ATOM =......FROM DATAFILE GRUB

Error N420 occurs during the input of datafile GRUB, if Programme GRIN cannot interpret one of the lines giving the Energy Variables for an ATOM. This error often occurs after faulty editing of Datafile GRUB. It is fatal, and you should therefore ask all Users if they have altered any records in GRUB.

22.2.36. N430-E

  (N430-E)  UNKNOWN MOLECULE NAME ... FOR MOLECULE ... OF SUBUNIT ..

This message only occurs with ATOM and not with HETATM records. (The difference should be clearly understood by the User. See Index under ATOM and HETATM). Message N430 is issued when the molecule name of an ATOM in the PDB file is not recognised; i.e. the molecule is not one of the 'Recognised Molecules' listed in datafile GRUB. Programme GRIN will then treat the ATOM as if it is in an 'Unknown Molecule'.

You should first check the molecule name of this ATOM, in case there is a misprint in the PDB file. Also check datafile GRUB in case it has been wrongly edited. Check in this User Manual to see if the molecule was included in datafile GRUB when that file was originally supplied. Please let us know at http://www.moldiscovery.com, if you think that your molecule should be added to Datafile GRUB for the next release of the Programmes.

Rerun GRIN if any mistakes have been found and corrected in PDB or GRUB.

On the other hand your molecule name may not occur in GRUB. In that case you should copy all the lines for the atoms of that particular molecule into another file. Edit that file, changing the first six characters on each line from 'ATOM ' into 'HETATM'. Process the file through Programme GREAT using the option: "Do you want to prepare a PDB file", in order to check that the names of all the HETAMS are acceptable "for use with Programme GRID".

Programme GREAT will give you an amended PDB file for the molecule with correct HETATM names. This amended PDB file for the molecule should be copied back to the end of the original PDB file, to give a modified PDB file. Finally remove the original ATOM records for the molecule from the modified PDB file (so that the molecule does not occur twice in the modified file, as both the original ATOM and the new HETATM records!!), and then reprocess the modified PDB file through Programme GRIN.

22.2.37. N440-I

  (N440-I)  A COMPLETELY EMPTY LINE NEAR MOLECULE...HAS BEEN DELETED

This message draws the user's attention to a blank line in the PDB input file.

22.2.38. N450-E

  (N450-E)  PARAMETER MAXNBR IS ONLY DIMENSIONED UPTO ....            
            NEIGHBOURS, AND THIS NUMBER HAS NOW BEEN REACHED          
            WHILE DEALING WITH HETERO-MOLECULE ........               
            PLEASE CHECK THE ELECTROSTATIC CHARGES FOR THAT           
            MOLECULE.

This error may occur while Programme GRIN is computing the charge distribution in a charged group in a hetero-molecule. The maximum number of HETATMs in the group is 20. If you want to increase this value, please contact us at http://www.moldiscovery.com .

22.2.39. N460-E

  (N460-E)  ERROR WHILE READING DATAFILE GRUB.                        
            TOO MANY ATOMS IN.....

Programme GRIN is dimensioned to deal with upto 200 atoms in each Recognised Molecule in file GRUB, and this limit has been exceeded. See below. This error is fatal.

22.2.40. N470-W

  (N470-W)  HETERO-MOLCULE .... APPARENTLY DOES NOT CONTAIN           
            ANY HETATMS.   PLEASE CHECK.

This uncommon error may occur, for example, if all the HETATMS in a hetero-molecule have been marked with a cross 'X' so that the next Programme GRID will ignore them. Examine the input PDB file to detect why something like this might have happened.

22.2.41. N480-W

  (N480-W)  ATOMS: ... IN MOLECULE ......                             
            AND:   ... IN MOLECULE ......                             
            ARE ONLY ... ANGSTROMS APART.    PLEASE CHECK.

This warning may be given if a water molecule is unexpectedly close to an atom of one of the other Target molecules. The User should decide if the reported position of the water is acceptable, before using Programme GRID.

22.2.42. N490-W

  (N490-W)  PLEASE CHECK THESE GUESSED VALUES:- ... ...

If Programme GRIN does not find suitable Energy Variables in datafile GRUB for one of the ATOMS in PDB, it may be forced to guess completely arbitrary values for that ATOM. Warning N490 is then printed so that you can take appropriet action.

The 'Unknown Molecule' procedure for selecting Energy Variables has failed in this case, because there is no ATOM with an appropriate name in datafile GRUB. These guesses are only a fall-back default procedure. It is most important for you to correct the guessed values, because the guesses may be quite inappropriate. See warning N510 below.

22.2.43. N500-I

  (N500-I)  PLEASE CHECK DIRECTIVE ALHY = ....                        
            WHICH SHOULD BE IN THE RANGE 0.01 TO 2.00

ALHY is the polarizability assigned to a hydrogen-bonding hydrogen atom. This directive will only be called for ATOMS if directive IHVA has been set. Programme GRIN will reject ALHY values outside the Range 0.009-2.0. If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.44. N510-W

  (N510-W)  PLEASE CHECK THESE SELECTED VALUES:-

If Programme GRIN does not find suitable Energy Variables in datafile GRUB for one of the atoms in PDB, it may try to find a similar atom in one of the other 'Recognised Molecule' types in GRUB. This is the 'Unknown Molecule' procedure. It is a default procedure, and the present message is printed if GRIN selects values in this way.

In many cases the Programme will not manage to find a suitable set of energy values, and there is a fall-back default procedure. This is described at message N490 above.

22.2.45. N520-W

  (N520-W)  SHOULD THERE REALLY BE A HEADER NEAR MOLECULE .......?

The HEADER should normally be at the top of the PDB file.

22.2.46. N530-W

  (N530-W)  RIGHT-SHIFTED OR RIGHT-EXTENDED ATOM NAME                 
            ... ... ... ... ...

This message may be issued if a line in the PDB file is shifted to the right, because a spurious character may then appear in the ALT column of the PDB format. The spurious character may suggest to Programme GRIN that the line in question is a duplicate atom entry.

A second possibility is that some of the atom names in your PDB file are too long. Only four characters are permitted for an atom name in PDB format: two for the atom symbol (eg SI for silicon) and two as identifiers (eg SI99 for silicon atom number 99). So if you have a silicon which is called "silicon 123" it must not be given the atom name SI123 because the 3 will appear in the column which is reserved in PDB format for the ALT indicator.

Also see message N860 below.

22.2.47. N540-E

  (N540-E)  ERROR AFTER INPUT OF HETATM...FROM DATAFILE GRUB

Error N540 occurs during the input of datafile GRUB, if Programme GRIN cannot interpret one of the lines giving the Energy Variables for a HETATM. This error often occurs after faulty editing of Datafile GRUB. It is fatal, and you should therefore ask all Users if they have altered any records in GRUB.

Note that error N540 reports the name of the last 'HET' atom which was input correctly. The error occurs on the next line of GRUB.

22.2.48. N550-E

  (N550-E)  ALL HETATMS MUST BE LISTED AFTER THE OTHER TARGET ATOMS.

File PDB normally starts with a HEADER, and it may then have a list of ATOM records. Any HETATM records in the file must be listed after all the ATOM records.

The list of ATOMS is optional and so is the list of HETATMS, although at least one of these lists must be present or there will not be any Target for Programme GRIN to process. However, there must not be any HETATM records mixed up with the ATOM list. The HETATMS, if any, must all follow after the ATOMS.

22.2.49. N560-E

  (N560-E)  ERROR DURING INPUT OF FIRST HETATM FROM DATAFILE GRUB

See error N540 above. This message occurs if the error occurs in the first HETATM record after the 'HET -1' line in datafile GRUB.

22.2.50. N570-W

  (N570-W)  PLEASE CHECK THIS INPUT LINE:                             
            ... ... ... ... ... ... ...

It sometimes happens that a line in the PDB file is misplaced to the right or the left, which can cause the data on that line to be misread. Message N570 is printed to draw attention to such a misplaced line, or to emphasise some other problem on the line of input.

22.2.51. N580-I

  (N580-I)  EXTRA HETATMS FOUND WITHIN COVALENT BONDING RANGE         
            NEAR HETATM ....   NEAREST HETATMS CHOSEN.

When Programme GRIN computes the positions of the hydrogens which are bonded to a HETATM of the Target molecule, it starts by identifying all the other HETATMS which are bonded to that HETATM. It will need to know the positions of some of these nearby HETATMS in order to calculate the hydrogen positions, but it sometimes identifies more HETATM neighbours than it actually needs. This message will then be printed for information, if the value of Directive LEVL > 4

A high value of Directive LEVL is required to force this message, because the finding of extra HETATMS does not constitute an error. However, this message may be useful for checking purposes.

22.2.52. N590-W

  (N590-W)  PLEASE CHECK THE FOLLOWING HYDROGEN COORDINATES WHICH     
            WERE COMPUTED FROM THE POSITIONS OF THE HEAVY ATOMS:      
            ..... ..... ..... .....

Programme GRIN computes the positions of hydrogen atoms. It does this on the basis of the ATOM or HETATM Type, which defines the hydrogen geometry. It then checks to see if the PDB input file contains a HETATM record for the same hydrogen.

If the hydrogen was already present in the original PDB file, then those hydrogen coordinates are normally used just as they are given in that PDB file. The computed coordinates are therefore rejected.

If the hydrogen was not present in the original PDB input, then the computed coordinates for the hydrogen are used.

Sometimes, however, there is a serious discrepence between the computed position for a hydrogen and the positions specified in the PDB input. In this case the present message N590 is printed to draw attention to the discrepence.

If this happens, the User should carefully check the coordinates, and find out what has happened. Perhaps the Type of a heavy ATOM or HETATM has been wrongly assigned; perhaps the molecule is severely strained so that the hydrogens are not in their expected positions; or perhaps there is a missing atom or abnormal bond-length or bond-angle, so that the subroutines in GRIN have not computed the hydrogen coordinates accurately.

Also see Warning N280 above.

22.2.53. N600-I

  (N600-I)  PLEASE CHECK DIRECTIVE NEHY = ....                        
            WHICH SHOULD BE IN THE RANGE 0.1 TO 9.0

NEHY is the effective number of electrons assigned to a hydrogen-bonding hydrogen atom. It influences the strenght of the Van der Waals interactions which the atom can make. This directive will only be called for ATOMS if directive IHVA has been set. Programme GRIN will reject EFHY values outside the Range 0.09- 9.5. If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.54. N610-W

  (N610-W)  PROBLEM COMPUTING THE CHARGE DISTRIBUTION IN              
            HETERO-MOLECULE ......    PLEASE CHECK THE                
            CHARGES OF THIS HETERO-MOLECULE

This message could be due to rounding errors when the electrostatic charge distribution of a large hetero-molecule was being calculated. It would be necessary to move to higher-precision accuracy in order to deal with the problem, which has not yet been reported. Please contact us at http://www.moldiscovery.com if you get this warning N610.

22.2.55. N620-E

  (N620-E)  THIS MOLECULE CONTAINS AN EXTRA ATOM: .....

This message is printed if a Recognised Molecule contains more ATOMS than the number specified in datafile GRUB. The PDB file should be checked to see if the molecule name, or the number of atom records in the moleule, is causing this error.

22.2.56. N630-I

  (N630-I)  THE PDB INPUT FILE HAS FINISHED WITH AN END RECORD

This message may be printed for information, if a set of ATOM records in the PDB file have finished with an END record. The use of an END record in this way may often terminate input, and any subsequent ATOM or HETATM records may then be lost.

END records should only be used at the end of the complete input file. They should not be inserted between individual molecules.

On the other hand, a TER record must always be used at the carboxy terminus of every protein sequence, in order to ensure that the carboxy group will be correctly included in the GRINKOUT file. Also see message N640 below.

22.2.57. N640-W

  (N640-W)  SHOULD THERE BE A TERMINAL OXT ATOM BEFORE THE            
            TER RECORD, AND AFTER ATOM ...  ?

A TER record usually comes after the terminal carboxy group of a protein sequence. The last amino-acid before the TER record should therefore contain a complete terminal carboxy group; i.e. it should contain an extra oxygen (OXT) atom as defined for PDB format.

This message is printed if the expected OXT atom is missing. Message N640 is only a warning and not an error, because the sequence may have ended with a blocked carboxy group or it may be a DNA sequence which does not have a carboxy terminus at all. Please check your file.

22.2.58. N650-W

  (N650-W)  SHOULD THERE BE A TER RECORD AFTER HETATM ...... ?

A TER record should only come at the end of a sequence of Recognised Residues, typically after the terminal carboxy group of a protein sequence. It should not be used after hetero-atoms.

22.2.59. N660-E

  (N660-E)  STILL MORE VALUES SELECTED, ASSUMED, OR GUESSED

22.2.60. N670-I

  (N670-I)  IF YOU DO NOT WANT SO MUCH LINEPRINTER OUTPUT,            
            PLEASE SET THE VALUE OF PARAMETER 'LEVL' TO A             
            SMALLER NUMBER; E.G: 2    SEE YOUR USER MANUAL

'LEVL' is one of the input parameters which control the running of Programme GRIN. It controls the amount of information which is printed to the lineprinter output file GRINLOUT, and LEVL is given a default value of 2 in the Programme. However, this value can be changed in the Command file"grin.in".

When LEVL is set equal to 4 a substantial amount of information is printed to the lineprinter output file GRINLOUT, which is useful for new Users. Quite soon, however, they may prefer to have less output in GRINLOUT, and message N670 reminds them that the value of LEVL can be reduced.

Look up LEVL in the index for more information if you are a new User.

22.2.61. N680-W

  (N680-W)  PLEASE CHECK CHARGES OF HETERO-MOLECULE .......

There are ionised groups in this hetero-molecule, and they bear unexpected charges. The User is advised to use an independent method for determining the charge distribution.

22.2.62. N690-W

  (N690-W)  THERE SHOULD BE AT LEAST ONE AMINO-ACID                   
            BEFORE A TER RECORD.

A TER record should only come at the end of a sequence of Recognised Residues, typically after the terminal carboxy group of a protein sequence. It should not come earlier in the sequence, or at the start of a PDB file.

If there are no Recognised Residues and no ATOM records, there should be no TER record at all. Hence there should be no TER record if the PDB file is all HETATMS.

22.2.63. N700-E

  (N700-E)  ERROR WHEN READING COORDINATE INPUT OR FILE LIST.         
            PROGRAMME GRIN HAS NOT FINISHED CORRECTLY.

Programme GRIN needs input from one or more PDB files. This input is read through channel INKO as described above. If the input through channel INKO is some coordinates in PDB format, then GRIN will process those coordinates. Alternatively the input to INKO may be a list of PDB filenames, and in that case Programme GRIN will process each of the PDB files named in the list, one after the other as a Set.

This error message N700 is given if the input to INKO does not seem to be an unequivocal set of PDB coordinates, nor an unequivocal list of PDB filenames. For example, you might have one PDB file with the file-name HETATM and another called PHENOL. Your FILE LIST might then have the word HETATM written on its first line. Programme GRIN would read this word and tentatively assume that it was reading a HETATM record in a file of HETATM coordinates. On reading the second line of the FILE LIST it would find the name PHENOL, and would then make further checks since this is not a recognised record name in PDB format. It would discover that a file called PHENOL existed, and would then issue this error message because it was confused!

Message N700 is also given if the PDB files which are named in the FILE LIST do not exist, or are not in the expected directory. See Set of Targets for more information.

22.2.64. N710-E

  (N710-E)  MORE THAN ... ATOMS TO BE STORED

The Programme is normally supplied with the number of atoms dimensioned upto 24000. If you want to increase this value, please contact us at http://www.moldiscovery.com .

This error is fatal.

22.2.65. N720-I

  (N720-I)  ATOM ... IN MOLECULE ... WILL BE TREATED AS AN            
            ETHER-TYPE OXYGEN.   PLEASE CHECK THE CHARGE              
            DISTRIBUTION IN THIS MOLECULE.

Programme GRIN has been determining the environment of an oxygen atom. This oxygen has two neighbouring atoms which are within covalent bonding distance, but a different Type of oxygen was expected. In these circumstances the oxygen will be treated as an ether oxygen. Typically this occurs when a sugar monomer cyclises, or forms a polysaccharide by combination with another sugar.

When sugar molecules cyclise or combine in this way, the electrostatic charge distribution in the molecule will alter. Programme GRIN can take account of this when dealing with the Recognised sugar molecules which were included in Datafile GRUB when that file was supplied by Molecular Discovery Limited. However, it cannot always deal with other cases, and this message will be printed if problems could have arisen.

If this information message N720 has been issued, the User should assess the electrostatic charge distribution of the Target before carrying out any GRID computations.

22.2.66. N730-W

  (N730-W)  UNEXPECTED ELECTRO-NEGATIVITY FOR HETATM ....             
            THE ELECTRO-NEGATIVITY HAS BEEN RESET TO 0.1

If an unreasonably large charge were assigned to a HETATM, its electronegativity might be very greatly reduced. In some cases this could lead to a nominally negative electro- negativity value, which has been reset to a small positive value by Programme GRIN. Warning N730 has been printed to let you know what has happened.

22.2.67. N740-I

  (N740-I)  TER RECORD MISSING BEFORE HETATMS.

HETATMS may occur in the PDB file after the last sequence of Recognised Residues has been input. That sequence would typically be a protein finishing with a carboxy terminal and a TER record. Message N740 is printed if the expected TER record is missing.

This message is only a warning, because the sequence may not end with a protein carboxy terminus; for instance it might be a nucleic acid. Please check your PDB file.

22.2.68. N750-W

  (N750-W)  UNEXPECTED ELECTRO-NEGATIVITY FOR HYDROGEN ATOM ....      
            THE ELECTRO-NEGATIVITY HAS BEEN RESET TO 0.1

If an unreasonably large charge were assigned to a hydrogen atom, its electronegativity might be very greatly reduced. In some cases this could lead to a nominally negative electronegativity value, which has been reset to a small positive value by Programme GRIN. Warning N750 has been printed to let you know what has happened.

22.2.69. N760-W

  (N760-W)  IT APPEARS THAT HETATM .......                            
            IS NOT COVALENTLY BONDED TO HETERO-MOLECULE ......        
            PLEASE CHECK THE CHARGES OF THIS HETERO-MOLECULE

If the Target were sodium acetate, for example, it would be wrong to treat this as one single molecule unless the sodium was close enough to the rest of the Target for covalent bonding to be significant. Such a system should normally be treated as two separate molecules. i.e. one sodium cation and one acetate anion. Different molecule names (ACID) or subunit identifiers (ISUB) or molecule numbers (NRES) should therefore be used for the two moieties sodium and acetate.

22.2.70. N770-I

  (N770-I)  ATOM ... IN MOLECULE ... WILL BE TREATED AS AN            
            NH AMIDE-TYPE NITROGEN

This message normally occurs when the amide nitrogen of an asparagine amino-acid side-chain has been glycosylated. It is therefore no longer an NH2 group, and its Energy Variables have been adjusted accordingly

22.2.71. N780-I

  (N780-I)  ATOM .... IN MOLECULE .... WILL BE TREATED AS AN          
            ETHER-TYPE OXYGEN

Some molecules can occur in more than one form. A prime example is provided by sugars, which can be represented as straight-chain molecules or as cyclic ethers. Moreover, sugars can polymerise into polysaccharides, and in both these cases an hydroxy oxygen will be changed into an ether-type oxygen.

Programme GRIN detects such changes by studying the geometry of any molecule containing an hydroxyl group, and assessing its relationships to other nearby molecules. If GRIN finds that an hydroxyl group has changed into an ether-link, the Energy Variables will be adjusted appropriately. This information message N780 will then be printed to the line- printer file GRINLOUT.

22.2.72. N790-W

  (N790-W)  TER RECORD MISSING BEFORE NTER

This message may occur when the Target contains more than one protein chain. Each chain should normally finish with a carboxy terminal OXT atom and a TER record. The start of the next chain at its N-terminal should then be indicated by the presence of an NTER record.

In this case an NTER record has been found without any immediately preceding TER record, and message N790 has been printed for information. It is not an error message, because the preceding sequence might have been a nucleic acid chain which does not have a carboxy terminus. Please check your file.

22.2.73. N800-E

  (N800-E)  PLEASE CHECK DIRECTIVE IHVA = ....                        
            WHICH SHOULD BE 0 OR 1

The value of directive IHVA determines whether LennardJones variables and an electrostatic charge will be assigned to the hydrogenbonding hydrogen atoms which are bonded to ATOMS. (These Energy Variables are always explicitly assigned to the hydrogens which are bonded to HETATMS). The default value of IHVA is zero, and error message N800 is printed if the User sets IHVA to any value except 0 or 1.

This error is fatal.

22.2.74. N810-W

  (N810-W)  MISSING ATOM .... IN MOLECULE ...

This occurs when a Recognised Molecule is being input. Programme GRIN knows what ATOMS should occur in every Recognised Molecule, because they are listed in datafile GRUB. Message N810 indicates that one of the expected atoms is missing from the PDB input file.

22.2.75. N820-W

  (N820-W)  PLEASE CHECK THESE ASSUMED VALUES                         
            ...... ...... .....

If Programme GRIN cannot find suitable Energy Variables in datafile GRUB for one of the HETATMS in PDB, it may be forced to assume complelely arbitrary values for that HETATM. This warning is then printed.

22.2.76. N830-W

  (N830-W)  THIS SEEMS TO BE A WATER RECORD   ...... ...... .....     
            PLEASE CHECK THE ATOM AND MOLECULE NAMES IN THE ORIGINAL  
            PDB FILE.   THE ATOM NAME WILL BE CHANGED TO ' OH2' AND  
            THE MOLECULE NAME TO 'HOH '

This message is given when a HETATM has the atom name: ' OHH' but its molecule name does not seem to be the name of a water molecule. See the note below about atom and molecule names.

The atom name: ' OHH' is explicitly reserved for HETATM waters, although other names (' OH2' and ' O2 ') may also be used. However, all three atoms of the water molecule must be explicitly given as three separate HETATM records in the PDB file, if you use the atom name ' OHH' for the oxygen of the water; i.e.: there must be one HETATM line for the oxygen atom and two more HETATM records for the two hydrogens of an ' OHH' HETATM water. Therefore:

If this is a HETATM oxygen atom of water, you should be sure that the molecule name in your GRINKOUT file is: 'HOH '.
If this is a HETATM oxygen atom of water, you must also make sure that all three atom records for water are present (ie: one oxygen and its two hydrogens), or you must use a different atom name for the oxygen. (The default atom name for the oxygen of a HETATM water is ' OH2' and this represents a complete water molecule; i.e. no extra hydrogen records are required for an ' OH2' water HETATM).
If this is not a water, then you must not use the ATOM name: ' OHH'.

Also see message N190 above.

Note on atom and molecule names: An ATOM or HETATM record contains both an atom and a molecule name. These are ATM(I) and ACID(I) in Format Statement 120 above (see under Format 120). They should not be confused with each other. This message N830 refers to both atom and molecule names.

22.2.77. N840-I

  (N840-I)  MOLECULE .... CONTAINS DEOXYRIBOSE AND THE ENERGY         
            VARIABLES OF C2* HAVE BEEN ADJUSTED ACCORDINGLY

Datafile GRUB contains Energy Variables for the atoms in adenosine and the other constituents of nucleic acids. These parameter values are provided on the assumption that the 2-hydroxy group of the ribose moiety is present. This message indicates that it is absent, and that Programme GRIN has adjusted the Energy Variables of ribose accordingly.

22.2.78. N850-W

  (N850-W)  TER RECORD MISSING AFTER ATOM .....

The PDB input file often consists of a list of ATOMS, optionally followed by a list of HETATMS. Alternatively it may consist of HETATMS alone. When ATOMS are present, however, the ATOM list should often be followed by a TER record.

Message N850 is issued if the TER record is missing, but does not always mean that there is an error. This TER record is simply to indicate that the end of a protein or nucleic acid chain has been reached. However, the TER record is essential for Programme GRIN if your Target is a normal protein, because this record tells the Programme that the sequence is to be terminated with a complete ionised carboxy group.

The PDB file should now be edited if your Target is a protein, to include the necessary TER record immediately after the terminal carboxy OXT oxygen and before the first HETATM. Also make sure that your PDB file does have the necessary extra C-terminal carboxy oxygen atom, and append this oxygen if need be. Make sure that it has the special atom name OXT as defined by PDB format. Then rerun Programme GRIN.

22.2.79. N860-E

  (N860-E)  RIGHT-SHIFTED HETATM NAME, OR DUPLICATE HETATM            
            ... ... ... ...

If the entry for a HETATM seems to appear twice in file PDB, the second appearance of the entry is copied to lineprinter file GRINLOUT with this warning. One entry or the other must be removed from the PDB file before re-running Programme GRIN.

This same error message may also be issued if a line in the PDB file is shifted to the right, because a spurious character may then appear in the ALT column of the PDB format. The spurious character will suggest to Programme GRIN that the line in question is a duplicate HETATM entry.

A third possibility is that some of the atom names in your PDB file are too long. Only four characters are permitted for an atom name in PDB format: two for the atom symbol (eg SI for silicon) and two as identifiers (eg SI99 for silicon atom number 99). So if you have a silicon which is called "silicon 123" it must not be given the atom name SI123 because the 3 will appear in the column which is reserved in PDB format for the ALT indicator.

Also see message N530 above and N980 below.

22.2.80. N870-E

  (N870-E)  SEARCHING FOR TOO MANY BONDED ATOMS NEAR ATOM ...

When Programme GRIN computes the positions of the hydrogen- bonding hydrogen atoms on an ATOM in the Target molecule, it starts by identifying all the other ATOMS which are bonded to that ATOM. This message will be printed if the required neighbours are not present. It often occurs if the Type of hydrogen bonding (N1IN) has been wrongly assigned, or if Subroutine NASIGN has been incautiously edited. Error N870 is fatal.

22.2.81. N880-E

  (N880-E)  MORE THAN .... ATOMS TO BE STORED

The number of atoms which can be processed by Programme GRIN is 24000. This value is usually large enough for all the ATOMS, HETATMS and the hydrogen atoms which are appended to the Target, but it has been exceeded in the present case; please contact us at http://www.moldiscovery.com .

This error is fatal.

22.2.82. N890-E

  (N890-E)  MAKE SURE THAT THE LAST SEQUENCE DOES                     
            FINISH PROPERLY WITH A TER OR AN END RECORD.

This warning may be printed if the PDB imput finishes abruptly. A TER or an END record may be needed in the PDB file. If you do not use one of these records, then the energy variables of hydrogen atoms may not be correctly assigned. Please check your file and add TER or END records if need be.

22.2.83. N900-I

  (N900-I)  PLEASE CHECK DIRECTIVE LEVL = ....                        
            AN INTEGER BETWEEN -2 AND 6 WAS EXPECTED

The value of directive LEVL determines the amount of information which is printed to the lineprinter file GRINLOUT. The default value is unity. Higher values give more information, and lower values give less. This message N900 is printed if the User sets LEVL to a value outside the range -1 to +6.

22.2.84. N910-W

  (N910-W)  CHECK CHARGES ON SULPHUR OF RESIDUE....OF SUBUNIT...

Programme GRIN checks for sulphur atoms in a protein which are close enough to form S-S bridges. When it finds them the atomic charges of the sulphur and its associated carbon (CB) atom are slightly adjusted from the normal values in file GRUB. This warning is printed if Programme GRIN cannot make these changes unequivocally.

22.2.85. N920-E

  (N920-E)  NO HETATMS FOUND.

When Programme GRIN computes the positions of the hydrogen atoms on a HETATM in the Target molecule, it starts by identifying all the other HETATMS which are bonded to that HETATM. If it tries to do this but finds that no HETATMS are present, this error message will be printed. This unusual error is fatal.

22.2.86. N930-W

  (N930-W)  ATOM ... IS NOT WITHIN THE HET LIST

When Programme GRIN computes the positions of the hydrogen atoms on a HETATM in the Target molecule, it starts by identifying all the other HETATMS which are bonded to that HETATM. If it tries to follow this procedure and finds that some of the bonded neighbours are not HETATMS, this error message will be printed.

A PDB input file for Programme GRIN may contain ATOMS; or it may contain HETATMS; or it may contain ATOMS followed by HETATMS. However the ATOMS must precede the HETATMS, and they must not be mixed in the file. Furthermore ATOMS and HETATMS must not be used together, in order to define different parts of the same molecule; ie a molecule may be made of ATOMS, or HETATMS but not both.

This Warning N930 often occurs when ATOMS and HETATMS have both been used for different parts of the same molecule.

22.2.87. N940-W

  (N940-W)  SUSPECTED ERROR IN NUMBER OF COVALENTLY                   
            BOUND NEIGHBOURS, OR THE NUMBER OF NEIGHBOURS             
            ONE PLACE REMOVED FROM ATOM  .......

When Programme GRIN computes the positions of the hydrogen- bonding hydrogen atoms on an ATOM in the Target molecule, it starts by identifying all the other ATOMS which are bonded to that ATOM. It will need to know the positions of some of these nearby ATOMS in order to calculate the hydrogen- positions, but it sometimes does not find the expected number of neighbours. This error message will then be printed.

Message N940 often indicates that the structure of the Target molecule is unusual, or that it is strained, or that it is a transition state. GRID is not calibrated for highly strained molecules or transition states, but we would be grateful at Molecular Discovery if can let us know when this message is given for a regular atom in a regular molecule.

Another possibility is that the structure is wrong, or that an error has been introduced into Datafile GRUB, or that the name of an ATOM is incorrect, or that the molecule name, number or chain identifiers have been misassigned. These possibilities should be checked.

22.2.88. N950-E

  (N950-E)  ERROR IN NUMBER OF HETATMS OR IN TOTAL                    
            NUMBER OF ATOMS COUNTED.

When Programme GRIN computes the positions of the hydrogen atoms on an ATOM or HETATM in the Target molecule, it starts by cataloguing all the other ATOMS and HETATMS. This error message will be printed if it does not find the expected number of ATOMS and HETATMS.

This message is fatal, and it should never occur. Please let us know at http://www.moldiscovery.com if you do get message N950.

22.2.89. N960-E

  (N960-E)  ILLEGAL H-BOND TYPE IN ATOM ...

The Type of hydrogen bonding on atoms and hetatms is defined by Type numbers. This message is printed if an unacceptable Type number has been used.

The Type of an atom is a very important concept for Programmes GRIN and GRID. See Index under Type for more information.

22.2.90. N970-W

  (N970-W)  SUSPECTED ERROR IN NUMBER OF COVALENTLY                   
            BOUND NEIGHBOURS, OR THE NUMBER OF NEIGHBOURS             
            ONE PLACE REMOVED FROM HETATM .......

When Programme GRIN computes the positions of the hydrogen atoms on a HETATM in the Target molecule, it starts by identifying all the other HETATMS which are bonded to that HETATM. It will need to know the positions of some of these nearby HETATMS in order to calculate the hydrogen- positions, but it sometimes does not find the expected number of neighbours. This error message will then be printed.

Message N970 often indicates that the structure of the Target molecule is unusual, or that it is strained, or that it is a transition state. GRID is not calibrated for highly strained molecules or transition states, but we would be grateful at Molecular Discovery if can let us know when this message is given for a regular atom in a regular molecule.

Another possibility is that the structure is wrong, or that an error has been introduced into Datafile GRUB, or that the name of a HETATM is incorrect, or that the molecule name, number or chain identifiers have been misassigned. These possibilities should be checked.

22.2.91. N980-W

  (N980-W)  DUPLICATE NAME                                            
            ... ... ... ... ...

If the entry for an ATOM seems to appear twice in file PDB, the second appearance of the entry is copied to lineprinter file GRINLOUT with this warning. One entry or the other must be removed from the PDB file before re-running Programme GRIN. Also see message N860 above.

22.2.92. N990-W

  (N990-W)  CHECK CARBOXY-TERMINAL OF RESIDUE.....OF SUBUNIT...

Programme GRIN attempts to detect the carboxy-terminal of each protein chain from the position of TER records. It then adjusts the atomic charges of the carboxy group and its neighbours, from the normal values in datafile GRUB. This warning may be printed if it cannot make the necessary changes unequivocally.

22.2.93. N1000-I

  (N1000-I) PLEASE CHECK DIRECTIVE QQHY = ....                        
            A VALUE BETWEEN -9.0 AND +9.0 WAS EXPECTED

The value of directive QQHY defines the electrostatic charge which will be assigned to hydrogen-bonding hydrogen atoms. This directive will only be called for ATOMS if directive IHVA has been set. Error message N1000 is printed if the User sets QQHY to a value outside the range -9.0 to +9.0. If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.94. N1010-W

  (N1010-W) INPUT ERROR AT LINE:- .....

This warning is printed while a PDB input file of Target coordinates is being read in. Unacceptable characters have been found at some point in an input line. For example letters may have been found where numbers were expected. The whole input line is copied to the lineprinter file GRINLOUT immediately after message N1010, and the approximate position of the error is underlined with asterisks.

22.2.95. N1020-W

  (N1020-W) PLEASE CHECK IF THERE ARE TOO MANY ATOMS WITHIN           
            BONDED DISTANCE OF OXYGEN ... IN MOLECULE ....?

Programme GRIN detects such changes by studying the geometry of any molecule containing an hydroxyl group, and its relationships to other nearby molecules. If it detects structural anomalies in the neighbourhood of the hydroxyl group, such as too many bonded neighbours, this message may be printed to the lineprinter file GRINLOUT as a warning.

22.2.96. N1030-I

  (N1030-I) TOTAL CHARGE ON ALL ATOMS, HETATMS AND HYDROGENS          
            WHICH ARE PART OF THE TARGET = ........

Generally speaking, the overall charge of the Target should be zero. One may sometimes want to study a charged Target, but this should only be done because of a deliberate decision made by the User. Message N1030 is provided so that you can check the overall charge of the Target, as defined by their GRINKOUT file, before starting any Grid runs.

Note that the charge in message N1030 is the charge of the Target in the GRINKOUT file produced by Programme GRIN. A User may have edited the GRINKOUT file, perhaps by putting crosses against some of the atoms (see under Cross X). If the GRINKOUT file has been edited, then the charge of the Target may also have been changed.

The charge of each molecule in the Target will be printed by message N1540 if directive LEVL is set to a high enough value. See message N1540 below.

22.2.97. N1040-E

  (N1040-E) UNACCEPTABLE INPUT FILE;                                  
            NO ATOM OR HETATM RECORDS

Programme GRIN expects an input file in Brookhaven PDB format, and this file must contain the coordinate positions of the atoms of the Target. Those coordinates must be defined by ATOM or HETATM records as described by the Brookhaven specification, and this error message will be issued if no ATOM or HETATM records are found.

Make sure that your coordinate input is an ASCII text file in Brookhaven format, and that it has at least one (!) ATOM or HETATM record. Also check your command file to be sure that the value of directives IDIR and INKO have not been changed to unacceptable values. Full information about Brookhaven PDB format can be obtained from:

The Protein Data Bank, Chemistry Department Brookhaven National Laboratory, Upton, NY 11973, USA

22.2.98. N1050-W

  (N1050-W) WHY ARE SO MANY ATOMS WITHIN BONDED DISTANCE OF           
            NITROGEN ... IN MOLECULE ...   ?

This message normally occurs when the amide nitrogen of an asparagine amino-acid side-chain might be glycosylated. Programme GRIN is making a search in order to find the bonded neighbours of this nitrogen, and has found too many neighbours for a reasonable structure. The User should inspect this part of the PDB input file, and may need to regularise some of the atomic coordinates.

22.2.99. N1060-W

  (N1060-W) UNEXPECTED CASE OF HYDROGEN GEOMETRY

Users should be fully aware of the concept of atom "Type" which defines several properties of the atom (or hetatm). One of these properties is the geometry of any hydrogens which are bonded to the atom in question. Each geometrical arrangement is called a "Case" and the Case is a sub-property of the Type. For example, Case 3 defines the arrangement of hydrogens in a methyl group.

The current Version of Programme GRIN provides ten standard Cases as described in this User Manual. The current version of datafile GRUB includes many more atom Types, but whenever one of these Types calls a Case, it is always one of the ten standard Cases.

The present error message arises when a Type attempts to call an undefined Case. This should only happen if a User has defined a new Type of atom in datafile GRUB .

22.2.100. N1070-E

  (N1070-E) 'HYD' IS A RESERVED NAME, AND MAY NOT BE                  
            USED IN THIS CONTEXT

The name 'HYD' must never appear as an atom or molecule name in any PDB file which is to be used as input to Grin and Grid. This requirement applies to both ATOM and HETATM records. It arises because the name 'HYD' is assigned to the hydrogen bonding hydrogens which are appended by Programme Grin to the original PDB input file. The assignment of these hydrogen-bonding hydrogens must be unique, and the name 'HYD' is reserved in order to ensure uniqueness.

If you have used the name HYD for a molecule in your PDB input file, you must rename the molecule before using that file as input for Programme Grin. For example, you might have used the molecule name 'HYD' because the Target itself was hydrocortisone. The name 'HYD' in the hydrocortisone file would have to be replaced by another name such as 'HYC', before using that file as input to Programme Grin. This replacement can usually be done globally by one or two keystrokes of your page editor.

22.2.101. N1090-I

  (N1090-I) YOUR PDB INPUT FILE HAD ... ATOMS WITH NEGATIVE           
            OCCUPANCIES.  THESE ATOMS WILL BE FORCED INTO THE         
            CORE OF THE TARGET IF IT'S REASONABLE TO DO SO.           
            GREAT CARE SHOULD BE TAKEN WHEN YOU TRY TO FORCE          
            ATOMS INTO OR OUT OF THE CORE OF THE TARGET.

The number in the occupancy field of a normal PDB record is zero or positive, but the User may change it to a negative number, and this negative number will be interpreted as a Flag by Programme GRIN. This Flag in the PDB file instructs Programme GRIN to force the atom into the Core of the Target. The atom's position will then be fixed during GRID runs, and it will not move when interacting with the Probe. It will be fixed, even if directive MOVE>0 in the GRID run.

Of course, the negative number might have got into the PDB file by mistake, and this information message is printed to remind the User what is going to happen with these particular atoms. He or she should check the PDB file, and should also check the GRINKOUT.DAT output file from GRIN. This is essential, because it is sometimes not possible to force atoms in the way one would like, the desired result may not have been obtained.

22.2.102. N1100-I

  (N1100-I) PLEASE CHECK DIRECTIVE VDHY = ....                        
            A VALUE BETWEEN 0.01 AND 2.0 WAS EXPECTED

The value of directive VDHY defines the Van der Waals radius which will be assigned to hydrogen-bonding hydrogen atoms. This directive will only be called for ATOMS if directive IHVA has been set. This message N1100 is printed if the User sets VDHY to a value outside the range 0.01 to 2.0 If you wish to alter these limits, please contact us at http://www.moldiscovery.com .

22.2.103. N1110-I

  (N1110-I) SULPHUR OF RESIDUE.....OF SUBUNIT...IS TREATED AS         
            A DISULPHIDE BRIDGE ATOM LINKED TO ....

This message is printed for information whenever a sulphur atom in a protein is treated as part of a disulphide bridge.

22.2.104. N1120-I

  (N1120-I) DIRECTIVE 'IHVA' HAS BEEN RESET EQUAL TO 1 FOR            
            THIS GRIN RUN, BECAUSE YOUR PDB INPUT FILE SEEMS          
            TO CONTAIN 'ATOM' RECORDS FOR HYDROGEN.

Traditional PDB files used Extended Atoms, in which any hydrogens were treated as if they were part of the heavy atom to which they were covalently bonded. Thus a methyl group would not have been treated as a cluster of one heavy and three separate hydrogen atoms, but as a single spherical Extended Atom.

When small molecules are being studied by Programme GRID, the atoms of the small molecule are normally represented by HETATM records, and every hydrogen in the molecule has its own HETATM record. Thus Extended Atoms are not used by GRID for small molecules.

When macromolecules are being studied with GRID one may either use traditional Extended Atoms, or may provide all the hydrogen coordinates as extra ATOM records (See Index under: Hydrogen ATOM Records). Directive IHVA must then be set, in order to ensure that each atom is assigned an appropriate size and charge. This message is to tell you that IHVA has been automatically set by Programme GRIN.

Please note that unrecognised atom names in ATOM records will be treated as hydrogens if they contain the character "H". For instance, the acetyl group (ACE) has a methyl group for its alpha-carbon, and this is called CA in Datafile GRUB. If this methyl is called "CH3" in your PDB file, it will not be identified as the CA moiety. Then by default it will be treated as a hydrogen because it contains the character "H", and information message N1120 will be issued.

22.2.105. N1130-E

  (N1130-E) OVERLAP ERROR.                                            
            PROGRAMME STOPS IN SUBROUTINE: NATMA2.

The number of atoms which can be processed by Programme GRIN is 24000.

This value must be large enough for all the ATOMS, HETATMS and the hydrogen atoms which are appended to the Target, and for all the Special Records which are written to GRINKOUT.DAT. However, it has been exceeded in the present case. If you want to increase this value, please contact us at http://www.moldiscovery.com .

This error is fatal.

22.2.106. N1140-W

  (N1140-W) PROBLEM WRITING THE LETTER H IN FRONT OF THE ATOM         
            SYMBOL ..... IN ORDER TO SHOW WHICH IS THE DONOR          
            HYDROGEN ON ATOM .... IN MOLECULE .....

The normal symbol for an atom or hetatm is a CHARACTER*4 variable ATM(I). However, as mentioned above, it is actually possible to use five characters for atom and hetatm names. The extra character preceeds the other four and is normally a blank. Alternativel it may be an 'H' if the atom is a hydrogen whose position has been computed by this Programme GRIN.

In this case the extra character position has already been used for the name of the heavy atom or hetatm itself. This has caused the present problem because the extra position is no longer available for the letter 'H'. Message N1140 will be printed whenever an abortive attempt is made to overwrite a five character atom or hetatm name with an 'H' in the first character position.

This message N1140 does not indicate an error, but the visual interpretation of the GRINKOUT output will be invalidated because the expected 'H' will be missing before some of the hydrogen-bonding hydrogen atoms. However, the next Programme GRID should accept such a GRINKOUT file and process it correctly.

22.2.107. N1150-E

  (N1150-E) MORE THAN .... ATOMS CANNOT BE STORED.                    
            PROGRAMME STOPS IN SUBROUTINE: NSTART

The number of Target atoms is 24000 in this Version of the Programmes. However the acceptable number of atoms is significantly less than this.

About 50 of the topmost registers are reserved for special purposes, and this reduces the acceptable number of atoms to about (MAXDIM-50). More registers are required to hold "Special Records" (which are marked by a plus sign "+" in column 6), and which describe the conformational flexibility of certain Target atoms. Other record lines may be marked with a cross "X" in column 6, and this shows that they have been rejected by the User or have been moved elsewhere in the file. Furthermore Programme GRIN will have computed the positions of an appropriate number of hydrogen atoms and added these to the file.

All these requirements tend to reduce the actual number of Target atoms which can be accepted by GRIN from the PDB input file, and there are too many atoms in your file. You can edit the file, perhaps removing unnecessary water molecules; please contact us at http://www.moldiscovery.com .

22.2.108. N1160-I

  (N1160-I) DIRECTIVE MOVE=1 BUT THIS HETATM WILL NOT BE              
            ALLOWED TO UNDERGO COMPLETE TORSIONAL ROTATION:           
   HETATM    8  C2  MOL     1       0.026  -1.722  -1.471             
            PLEASE CHECK IF THIS IS WHAT YOU WANT.

A search is being made for atoms outside the core of the Target; ie: for atoms whose position may change in response to the Probe. This particular atom (or HETATM) is not obviously in the core, but a GRINKOUT file is being prepared in which its motion is restricted.

HETATM C2 is the carbon of a methylene group, and it might be linked to a benzene ring in the core like this:

GRIN would assign some double-bond character to the bond (ab) from the benzene to the oxygen if this was a short bond. In that case GRIN would not allow torsional rotation of C2 around the ab bond, and information message N1160 is issued in order to let you know what has happened.

22.2.109. N1170-I

  (N1170-I) DIRECTIVE MOVE=1.                                         
            THIS ATOM WILL BE ALLOWED TO UNDERGO FULL                 
            TORSIONAL ROTATION:                                       
   ATOM     98  CD  LYS    15       0.026  -1.722  -1.471             
            PLEASE CHECK IF THIS IS WHAT YOU WANT.

A search is being made for atoms outside the core of the Target; ie: for atoms whose position may change in response to the Probe. It may not be immediately obvious that this particular atom (or HETATM) is outside the core. However, a GRINKOUT file will be prepared in which it is allowed to rotate round its linker atom.

In the following example CD is a methylene group in a Lysine side chain, and it is bonded to the CG methylene group like this:

                       a   b
               CORE--CB-----CG            NH
                             \           /
                              \         /
                               CD------CE

GRIN is treating the CB and CG methylene groups as being in the Core of the protein, while CD is outside the Core. GRIN is therefore going to allow torsional rotation of CD around the ab axis, and information message N1170 is therefore issued in order issued in order to let you know what has happened.

22.2.110. N1180-E

  (N1180-E) MORE THAN....ATOMS, INCLUDING HYDROGENS, TO BE STORED.

The number of atoms which can be processed by Programme GRIN is 24000.

This value is usually large enough for all the ATOMS, HETATMS and the hydrogen atoms which are appended to the Target, but it has been exceeded in the present case. There are too many hydrogens; if you want to increase this value please contact us at http://www.moldiscovery.com .

22.2.111. N1190-I

  (N1190-I) YOUR PDB INPUT FILE HAD ... ATOMS WITH NEGATIVE           
            B VALUES.   THESE ATOMS WILL BE FORCED OUT OF THE         
            CORE OF THE TARGET IF IT'S REASONABLE TO DO SO.           
            GREAT CARE SHOULD BE TAKEN WHEN YOU TRY TO FORCE          
            ATOMS INTO OR OUT OF THE CORE OF THE TARGET.

The number in the BVAL field of a normal PDB record is zero or positive, but the User may change it to a negative number, and this negative number will be interpreted as a Flag by Programme GRIN. This Flag in the PDB file instructs Programme GRIN to force the atom out of the Core of the Target. The atom's position will then be allowed to move when it is interacting with the Probe, and when Directive MOVE>0 in Programme GRID.

22.2.112. N1200-W

  (N1200-W) YOUR PDB INPUT FILE HAD ... ATOMS WITH BOTH A             
            NEGATIVE OCCUPANCY AND A NEGATIVE B VALUE!!               
            ALL THESE PAIRED VALUES WILL BE IGNORED.                  
            GREAT CARE SHOULD BE TAKEN WHEN YOU TRY TO FORCE          
            ATOMS INTO OR OUT OF THE CORE OF THE TARGET.

(N1200-W) YOUR PDB INPUT FILE HAD ... ATOMS WITH BOTH A NEGATIVE OCCUPANCY AND A NEGATIVE B VALUE!! ALL THESE PAIRED VALUES WILL BE IGNORED. GREAT CARE SHOULD BE TAKEN WHEN YOU TRY TO FORCE ATOMS INTO OR OUT OF THE CORE OF THE TARGET. The number in the occupancy field of a normal PDB record is zero or positive, but the User may change it to a negative number and it will then be interpreted as a Flag by Programme Grin. This Flag instructs the Programme to force the atom into the Core of the Target.

In just the same way the number in the BVAL field may be made negative, and this will instruct the Programme to force the atom out of the Core. However, these two flags are mutually incompatible, and should not be used together for the same atom. This message N1200 is a warning that both flags have been set. You should check your PDB file and correct the mistake.

22.2.113. N1210-E

  (N1210-E) MORE THAN....ATOMS CANNOT BE STORED

22.2.114. N1220-W

  (N1220-W) PLEASE CHECK IF THIS ATOM IS A HYDROGEN:                  
            ... ... ... ...                                           
            IT SEEMS TO HAVE AN UNUSUAL BOND LENGTH

This atom has been identified as a hydrogen; perhaps it has a hydrogen-like name including the letter H. However hydrogens usually make one covalent bond of about one Angstrom length, and this particular atom does not seem to make such a bond. Please check your file.

You will get this warning if the bond length to the hydrogen is outside the range 0.4 - 1.3 Angstrom. Please let us know at http://www.moldiscovery.com if you cannot find the reason why you are getting this message.

22.2.115. N1230-W

  (N1230-W) UNEXPECTED LENGTHS FOR SOME BONDS, SO THAT                
            SOME ATOMS MAY NOT HAVE BEEN CORRECTLY                    
            ASSIGNED IN THE CORE OF THE TARGET.                       
            PLEASE CHECK LENGTHS

Programme GRIN assigns atoms to the Core of the Target by checking the lengths of covalent bonds and hydrogen bonds and non-bonded contacts between atoms. This warning N1230 was issued because GRIN has not been able to assign atoms unequivocally to the Core.

This may reflect an unusual feature of your molecule. We suggest that you start by checking the conformation as specified in your PDB file. Perhaps there is a side-chain which is making close Van der Waals or hydrogen-bond contacts with the Core, and GRIN cannot establish whether it should be treated as a flexible side-chain, or as a fixed part of the Core.

After checking the PDB file you can edit it, and use negative OCCUP or BVAL assignments in order to ensure that the atoms are assigned correctly to the Core or to a flexible region.

22.2.116. N1240-E

  (N1240-E) CANNOT DEAL WITH THIS EXTRA ATOM:                         
            ... ... ... ...                                           
            PLEASE CHECK ITS ATOM NAME AND COORDINATES

This warning is normally issued in connection with a hydrogen record. The hydrogen may be more than 2.0 Angstrom from the nearest heavy atom, which is too long for a covalent bond. Alternatively, the hydrogen may not be in any molecule at all, perhaps because its molecule name or number or chain indicator are wrongly defined in the PDB file.

Another possibility is that the alternate position indicator of the hydrogen record has been set. This may be because the hydrogen has been given an atom name which is too long for PDB format. Only four characters are permitted for an atom name in PDB format: two for the atom symbol (eg SI for silicon) and two as identifiers (eg SI99 for silicon atom number 99). So if you have a hydrogen which is called "hydrogen 123" it must not be given the atom name H123 because the 3 will appear in the column which is reserved in PDB format for the ALT indicator.

22.2.117. N1260-I

  (N1260-I) ATOM ..... IS MAGNESIUM IN THE LIST OF DNA COUNTER-       
            IONS, AND REPLACES A PAIR OF POTASSIA WHICH WOULD         
            HAVE BEEN TOO CLOSE TO EACH OTHER.

Potassium counter-ions are normally added to DNA by Programme Grin, in order to balance the anionic charge of the phosphate groups of DNA. It sometimes happens that two potassia have been placed unacceptably close to each other, and then one magnesium may be used instead. This helps to maintain the electrostatic charge balance, without crowding two mono-cations very close together.

22.2.118. N1280-I

  (N1280-I) ATOM ..... HAS BEEN CROSSED OFF THE LIST                  
            OF DNA COUNTER-IONS BECAUSE IT WOULD BE                   
            TOO CLOSE TO ANOTHER MOLECULE ALREADY                     
            LIGANDED TO THE DNA

DNA Targets (and RNA) have phosphate backbones, and the massive negative charge of these phosphates would overwhelm everything else in the Grid Force Field in the absence of appropriate counter-cations. Programme GRIN therefore adds potassium (or magnesium) counter-ions to a DNA Target in order to neutralise the overall negative charge. The counter-ions are placed well into the water phase, at 10 Angstrom distance away from the nearest phosphate phosphorus atom.

This information message N1280 has been issued because GRIN has found some other atoms at the place where it would like to put a counter-ion. These other atoms could come, for example, from a repressor protein already liganded to the DNA. However, GRIN cannot put a counter-ion on top of another Target atom, and so it has crossed this particular counter-ion off the list of Target atoms in the GRINKOUT file.

It often happens that the repressor protein has a positive charge, and the loss of a potassium counter-ion is the appropriate adjustment. See above for more information, under the heading NUCLEIC ACID COUNTER IONS.

22.2.119. N1310-W

  (N1310-W) INPUT ERROR AT LINE                                       
            ...........................

This error occurs if Programme GRIN cannot interpret the characters on an input line from file PDB. See error N1010 above.

22.2.120. N1420-I

  (N1420-I) YOU MAY WANT TO PROTONATE SOME OF THESE GROUPS IN         
            ORDER TO REDUCE THE OVERALL CHARGE OF THE TARGET:

Yours Target is not electrically neutral, and this will inevitably influence the results from Programme Grid whenever you use a charged Probe. In these circumstances you may want to reduce the overall anionic charge of the Target, perhaps to simulate its condition in vivo. Programme Grin has identified some anionic groups (e.g.: aspartic acid side chains) whose location in the Target suggests that they might well be protonated. Also see message N1620 below.

22.2.121. N1430-W

  (N1430-W) THE TARGET HAS A SURPRISINGLY SMALL CORE.                 
            PLEASE USE NEGATIVE OCCUP SETTINGS IN ORDER TO            
            GET A CORE OF AT LEAST 7 ATOMS, OR USE NEGATIVE           
            BVAL SETTINGS TO ELIMINATE THE CORE COMPLETELY.

Some molecules have a small cluster of atoms which might or might not be treated as a Core. For example the ring and first methylene group of 3-pentyl-furan are rigid:

                     C===C---CH2.CH2.CH2.CH2.CH3
                    /    |
                   O     |
                    \    |
                     C===C

but these six heavy atoms would make a very small core. GRIN has therefore issued this message so that you can decide whether you want to process this molecule as:

A rigid core of six heavy atoms with a flexible CH2.CH2.CH2.CH3 side chain, or
A traditional Target for GRID in which the positions of all the atoms are fixed.

You can now influence this decision by resetting the OCCUP and/or BVAL parameters for each atom to a negative value in your PDB file, and then rerunning GRIN. This is described in detail above, under the heading FORCING ATOMS INTO THE CORE. Bear in mind, however, that you cannot make all the atoms of this molecule "flexible", because the torsion angles of the furan ring itself cannot rotate freely.

22.2.122. N1440-W

  (N1440-W) PLEASE CHECK THE PROTONATION OF RESIDUE ... ...           
            THE NAME OF THE RESIDUE (HIS,HID,HIE,HIP OR HI0)          
            IN YOUR PDB FILE MAY NOT CORRESPOND TO THE NUMBER         
            OF HYDROGENS BONDED TO SIDE-CAHIN NITROGEN ATOMS.         
            COMPARE YOUR PDB FILE WITH DATAFILE GRUB.

Your PDB input file contains ATOM records for Recognised Residues, and this residue is a histidine. There are two nitrogens in the histidine side-chain, each of which may either have a lone pair, or may have a bonded hydrogen. The electrostatic charge of the side-chain may therefore be either -1 or 0 or +1.

A normal histidine will have a hydrogen attached to one of the side-chain nitrogens, while the other nitrogen will have a lone pair. It will be uncharged, and you should use the residue name HIS for this normal histidine. Programme GRID will then try both hydrogen positions, simulating a tautomeric equilibrium.

Other options are available. For instance if neither nitrogen has a hydrogen you should use the residue name HI0. If both have a hydrogen use HID. The various options are shown in datafile GRUB.

Message N1440 is only issued when your PDB file contains ATOM records for the hydrogens of the histidine. It is then important that the residue name in your PDB file corresponds to the number of hydrogen records. For instance, it would be wrong to use the name HI0 if there was an ATOM record for a hydrogen bonded to one of the side-chain nitrogens.

You should now decide on the protonation state which you want for this histidine side-chain. Next decide which residue name should be used, by studying the options in Datafile GRUB. Then edit your PDB input file, giving the histidine the appropriate name (eg HIS or HID or HIP etc), and:

either have no ATOM records for any of the hydrogens of that histidine, in which case GRID will use the extended-atom approach, or
have all the hydrogens of that histidine shown by individual ATOM records, making certain that the correct nitrogens are protonated to correspond with the chosen residue name, or
have just the polar hydrogens of that histidine shown by individual ATOM records, again making certain that the correct nitrogens are protonated to correspond with the chosen residue name.

22.2.123. N1490-W

  (N1490-W) PLEASE CHECK THE NUMBERS OF POLAR AND                     
            NON-POLAR HYDROGENS.

Programme GRIN recognises the distinction between ATOM and HETATM hydrogens, and this message applies to ATOM hydrogens in particular. This warning may be issued if some of the ATOM hydrogens of your Target are defined by separate ATOM records in your PDB file, but other ATOM hydrogens are not.

Two situations are straightforward:

The User provides records for all Target hydrogens,
The User provides no hydrogen records,

and warning N1490 will not be issued in either of these cases. In practice, however, there are plenty of intermediate situations. Is the hydrogen of an SH group polar or non-polar? Have all three hydrogens of an N-terminal nitrogen been provided? Has the User provided all the polar and none of the non-polar hydrogens. .... and so on.

Message N1490 is a "fail-safe" warning which is issued if GRIN has detected a possible inconsistency in the way in which the hydrogen ATOMS have been provided in the PDB file. It most commonly occurs if the polar (ie hydrogen-bonding) hydrogen ATOMS are given and the non-polar hydrogen ATOMS are omitted from the PDB file. Message N1490 is not fatal, but we recommend that you reconsider the way in which the hydrogen ATOMS are listed.

This message will be suppressed if directive LEVL is less than 3.

22.2.124. N1500-E

  (N1500-E) PLEASE CHECK DIRECTIVE MOVE=.....                         
            THE VALUE OF THIS DIRECTIVE MUST BE 0 OR 1

MOVE is a directive in both Programme GRIN and GRID. It can take the values 0 and 1 in Programme GRIN, but no other values are allowed in GRIN. See the instructions for Directive MOVE in Programme GRIN.

22.2.125. N1510-E

  (N1510-E) PROBLEM WITH:                                             
            ... ... ... ... ...                                       
            CANNOT FIND OR CANNOT READ THIS FILE

Programme GRIN is processing several PDB input files one after the other as a Set. The named file is listed in the FILE LIST, but either it does not exist in the expected directory, or it cannot be read as a PDB input file.

Check first to be sure that the file does exist in the expected directory. Then display it on the VDU screen, in order to make sure that it really is a correct uncorrupted PDB file. Finally check the file permissions to be sure that Programme GRIN is not prevented from reading the file due to some permission mis-match

22.2.126. N1530-W

  (N1530-W) PLEASE CHECK THE NUMBER OF HYDROGENS BONDED               
            TO ATOM ... ... ... IN MOLECULE ... ... ...

Part of your Target is composed of ATOMS in Recognised Molecules, and some of these ATOMS may be hydrogens. There is a column in Datafile Grub which is used in order to check the number of these hydrogens in your PDB file. This column in GRUB lists the expected number of hydrogens which should be associeted with each heavy ATOM. (For example the number would be zero for a protein backbone carbonyl oxygen, and would be 1 for an alpha carbon.).

Programme GRIN then expects that there will be either:

No ATOM records for any of these expected hydrogens in your PDB file, or
One ATOM record for each expected hydrogen.

Warning N1530 is normally given because some ATOM hydrogens are provided in the PDB file, but the number of hydrogens does not tally with the expected number as defined in Datafile GRUB.

There are a number of special situations: eg N-terminal nitrogens; histidines either neutral or charged; neutral or ionised carboxyl groups; hydroxyl groups in sugars and in DNA and RNA. In fact, if some ATOM hydrogens are given in the PDB file but other hydrogens are missing, there is a vast number of combinations which must be considered. A fail-safe approach has therefore been followed, and this message may sometime be given when it is not actually required.

Warning N1530 does not deal with HETATM hydrogens. It is only issued if directive LEVL is greater than 1. Smaller values of LEVL give message N1590.

22.2.127. N1540-I

  (N1540-I) THE TOTAL CHARGE OF HETERO-MOLECULE: ....... IS ....

This message will be printed if parameter LEVL is set to more than 2 during the GRIN run. The overall charge assigned to each molecule of the Target can then be examined by the User. The electrostatic charge of a molecule is normally a whole number, and this can be checked.

22.2.128. N1590-W

  (N1590-W) PLEASE CHECK THE NUMBER OF HYDROGENS                      
            IN MOLECULE ... ... ... ...

This message will be printed if parameter LEVL is set to less than 2 during the GRIN run. It is issued once for each molecule. More information can be obtained by resetting LEVL to a higher value. Message N1530 will then be issued (see above) with details about each offending ATOM.