HBPLUS manual v.3.06
CONTENTS ======== 1 How to Use HBPLUS - Quick Instructions 2 How to Use HBPLUS - Full Instructions 2.1 Installation 2.2 Glossary of terms used in this guide, and in the program 2.3 The Command-Line Options 2.4 Input Files 2.5 Output File(s) Format 2.6 Adding New Residue Types 3 The Science 3.1 Introduction to Hydrogen Bonds 3.2 The Algorithm 3.2.1 Calculation of the Hydrogen Positions 18.104.22.168 Hydrogens Bound to sp2 Hybridised (Trigonal Planar) Donors 22.214.171.124 Hydrogens Bound to sp3 Hybridised (Tetrahedral) Donors 3.3 Selecting Potential Hydrogen Bonds 3.4 Orienting ASN, GLN and HIS side-chains 3.4.1 Introduction 3.4.2 The Algorithm 4 References SECTION 1. HOW TO USE HBPLUS - QUICK INSTRUCTIONS ================================================= hbplus.exe [options] [cleaned filename] [uncleaned filename] HBPLUS inputs a Brookhaven Protein Database (Bernstein et al 1977) format file, and outputs a list of potential hydrogen bonds. The interactions that qualify as hydrogen bonds must be between listed donor and acceptor atoms, and have acceptable geometries. A series of command-line options can be used to change these criteria, output a PDB-format file including generated hydrogen positions and extend the hydrogen bonding atoms, amongst other possibilities. The program is sensitive to relatively trivial mistakes in Brookhaven files, and it is stronly recommended that you run a program to check and correct the Brookhaven file before running HBPLUS. Such a program, "Clean", by DK Smith, R Laskowski and G Hutchinson, is distributed with HBPLUS subject to the same conditions of use. If HBPLUS is already installed, then to run it simply type . . . example% hbplus /idata/new/p1mbd.new /data/pdb/p1mbd.pdb The following output should be produced . . . HBPLUS Hydrogen Bond Calculator v 2.25 Jan 22 14:54:13 GMT 1994 (c) I McDonald, D Naylor, D Jones and J Thornton 1993 All Rights Reserved. Configured for 60000 atoms and 20000 residues. Criteria Minimum Angles; DHA 90.00, HAAA 90.00, DAAA 90.00 Maximum Distances; D-A 3.9, H-A 2.5, S-S 3.0 Maximum angles at aromatic acceptors DAAX 20.00, HAAAX 20.00 Minimum covalent separation 3 Covalent bonds Processing "/idata/new/p1mbd.new" Reading PDB file "/data/pdb/p1mbd.pdb" for CONECTs . . . PDB file contained 50 CONECT records 1653 atoms selected from 541 residues. 50 CONRECS used. Adding Polar Hydrogens. NOTE: 1MBD/-0093-HIS NE2 forms 3 covalent bonds. Checking for disulphide bridges . . . 0 disulphide bonds found. Opened output file "p1mbd.hb2". Checking for hydrogen bonds . . . 1244 hydrogen bonds found. [end of output] Your file of hydrogen bonds should be called p1mbd.hb2, and lie in the current directory. If HBPLUS is not already installed in your machine, installion instructions can be found in section 2.1. SECTION 2. HOW TO USE HBPLUS - FULL INSTRUCTIONS ================================================ 2.1 Installation You should be an academic user and have sent a signed confidentiality agreement to the authors (address at the end of the instructions). If you do not have a copy of HBPLUS and would like to receive one (free to academic users), please detach the confidentiality agreement from the end of this document, sign it, and send to the address given. Please allow other people in your department to use your copy of HBPLUS, but do not allow them to make their own copy. HBPLUS is now distributed with two other programs, "Clean" by DK Smith, R Laskowski and G Hutchinson, and "Access" by S Hubbard. "Clean" is recommended for use on brookhaven files before HBPLUS is used on them. "Access" is included so that the tools for the algorithm for analysis of Asn, Gln and His side-chains that is built into HBPLUSv3.0 can be fully exploited. The package also includes a simple C Shell script, "chkqnh", that calls "Clean", "Access" and "HBPLUS" to provide an analysis of the hydrogen bonding around Asn, Gln and His side-chains. HBPLUS is available by anonymous ftp as a 'crypt'ed file, hbplus.tar.Z.cr. On a unix system, use unix> crypt [password] < hbplus.tar.Z.cr >! hbplus.tar.Z unix> uncompress hbplus.tar.Z unix> tar xf hbplus.tar unix> make The password changes regularly to prevent people who have not signed the confidentiality agreement form getting copies - the current password will be emailed to you when we receive the document. If you have problems compiling HBPLUS using 'make', then edit 'Makefile'. A couple of lines in the Makefile are preceded by '#' - these lines are ignored by 'make'. Some lines have suggested alternatives next to them in the file, preceded by '#'. If none of these options seem appropriate, or they don't work, then email firstname.lastname@example.org, and I'll try to work out why. Most compilers generate one or more warnings about the source code, but no errors. HBPLUS has been compiled and run on UNIX systems and VAX/VMS but proved too large for some IBM PC clones. It should be quite simple to arrange the system to call the executable file. On VAX/VMS, I compile with "cc hbp_gen.c" "cc hbp_inpdb.c" "cc hbp_findh.c" "cc hbp_hhb.c" "cc hbp_qnh.c" "cc hbp_main.c" "link /executable=hbplus.exe hbp_inpdb.obj,hbp_hhb.obj,hbp_qnh.obj,hbp_main.obj" The .com script to analyse Asn, Gln and His side-chains, using both access and brkcln, has not yet been implemented for VAX/VMS. If you are on a VAX or VMS and want to run access and clean before HBPLUS in order to analyse Asn and Gln side-chains, then use these lines. "fortran clean" "link /executable=clean.exe clean.obj" "fortran asurf" "link /executable=asurf.exe asurf.obj" If you want to use command-line parameters, you must execute "HBPLUS :== $DRIVE[DIRECTORY.NAME]HBPLUS.EXE" after compilation, preferably placing it in your login.com file. 2.2 Glossary of terms used in this guide, and in the program. Atoms are described by their position relative to the hydrogen bonds. Fig 1: abbreviations used for atoms round H-bonds DD1 AA1 DDD D \ / \ / \ D--H::A DD H::A / \ \ DD2 AA2 AA Legend -- Covalent Bond H Hydrogen DD1,DD2 Donor Antecedents :: Hydrogen Bond D Donor AA1,AA2 Acceptor Antecednets A Acceptor Atom Names Througout this document, and in the output files, atoms are named with the four-letter atom codes used in the Brookhaven database. The first two letters specify the element name, eg C, N, O, CU etc. The third digit is the greek letter remoteness code translated as A, B, G, D, E, Z, H instead of alpha, beta, gamma, delta, epsilon, zeta, eta. The fourth digit is a numeric branch designator. For instance the two side-chain oxygens of glutamate are labelled "GLU OE1" and "GLU OE2". A hydrogen atom is specified slightly differently. The third and fourth digits are the same as for the atom it is attached to, the second digit is the chemical symbol and the first digit is an additional branch designator. For instance, the hydrogens on the side-chain nitrogen of a glutamine are labelled "GLN1HE2" and "GLN2HE2". 2.3 The Command-Line Options Most of these options can be combined together, eg -IxLo, but those options that expect an argument must be solitary (eg -a 60.0) or at the end of a "set" (eg -ILoxa 60.0). The most oft-used command-line options are '-O' (to give a PDB format file that includes all polar hydrogens) and '-Q' (to analyse the preferred orientations of Asn, Gln and His side-chains). -a The next argument is the minimum angle. This option sets the minimum D-H-A, H-A-AA and D-A-AA angles (Default 90.0 degrees). See below for the definition of these angles. -A The next three arguments are the minimum D-H-A, H-A-AA and D-A-AA angles, respectively. -b The next argument is the maximum angle with the perpendicular for amino-aromatic hydrogen bonds (Default 20.0 degrees). This only matters if the "R" option has been used to switch aromatic hydrogen bonds on. -B The next two arguments are the maximum H-A-Perpendicular and D-A-Perpendicular, respectively, where "Perpendicular" is the line perpendicular to the plane of an aromatic acceptor running throught the putative acceptor. -c/C In the output, change CYS SG to CSS SG or CYH SG, the former indicating that the sulphur is involved in a disulphide bridge. (The default is to refer to all cysteines and cystines as CYS). -d/D The next argument is the maximum D-A distance, in Angstroms (Default 3.9 Angstroms). -e/E This is used in the form "-e/E atomname number". The number of hydrogen bonds that atom "atomname" is theoretically able to donate ("E") or accept ("e") is set to "number". Although it rarely makes practical difference in this release of HBPLUS whether an atom is declared to be able to accept 1, 2 or 3 hydrogen bonds, including the number is still obligatory. It DOES make a difference to donors, as it is also the number of hydrogens to add to the *.h structure output with the "o" option. The setting "-1" is used for aromatic acceptors. The format of "atomname" is the same as in the output files and must be exactly seven characters long. Preceding and trailing spaces must be included. For example "-e 'MET SG ' 0" redeclares the methionine sulphur as incapable of hydrogen bonding, and "-e 'PRO N ' 1" declares the proline nitrogen as a hydrogen bond acceptor. See section 2.6. -f The next argument names a file of command-line options. The syntax of this file is rather like the UNIX shell - "#" introduces comments and the three quote types "&dquot;", "`" and "'" are used, but escape characters are not recognised. -h/H The next argument is the maximum H-A distance, in Angstroms (Default 2.5 Angstroms). -K Use Kabsch-Sander positions for the hydrogens (ie with the NH bond parralel to the preceding CO bond) rather than the Pauling position (bisecting CA-N-C). -k Use the Pauling position (default). -M The next two arguments are a residue name and a list of atoms to be added to that residue's list of included atoms. See section 2.6 -N Generate a list of neighbours, rather than hydrogen bonds. Covalently bonded and nearly-bonded contacts are excluded, but all other contacts within the maximum D-A distance are listed. The output file suffix changes from .hb2 to .nb2 [or, if used with the -L options, from .hhb to .nnb]. -n Disables "neighbours" option. Generate the list of hydrogen bonds. As you may have gathered, this is the default. -o/O Output a *.h file of atomic co-ordinates that include hydrogens. The format is an abbreviation of the Brookhaven data file. (Default is not to do this.) -P Output a list of all donors and acceptors. Aromatic acceptors can accept "-1 H-Bonds". -q Input a .asa file output by Simon Hubbard's program ACCESS (1992,4). HBPLUS looks for a file in the current directory that has the same name as the brookhaven file, but the .asa suffix. [Version 3.0 onwards] -Q Input a .asa file from ACCESS, and investigate the H-bonding patterns of ASN, GLN and HIS side-chains, producing a listing of H-bonds by side-chain and atom accessibilities, and classifying the conformation in the PDB file relative to the alternative as "Highly Suspect", "Slightly Suspect", "Indifferent", "Slightly Optimal" or "Highly Optimal", depending on which conformation further satisfied hydrogen bonding potential. This automatically triggers the '-X' option. [Version 3.0 onwards] -R Allow atoms in the aromatic rings of Tyr, Trp and Phe to accept amino-aromatic hydrogen bonds. -r Disables amino-aromatic hydrogen bonds (the default). -s/S The next argument is the cutoff distance for assigning a disulphide bridge. (Default 3.0 Angstroms) -T The next argument is a residue and the subsequent argument is a list of covalent bonds formed within that residue. See section 2.6. -u The next argument is a residue to be added to the HBPLUS residue list. See section 2.6 -U The next argument is a residue that is predefined as having all the atoms, covalent bonds and hydrogen bond donors and acceptors of the residue named in the subsequent argument. See section 2.6 -v/V The next argument is the number of covalent bonds that count as nearly bonded. Contacts that are "nearly bonded" do not count as hydrogen bonded. The default is two. -x Exchange the side-chains of Histidine, Glutamine and Asparagnine. These side-chains are difficult to resolve crystallographically with certainty, which is why there is the option of adding potential hydrogen bonds that would be formed if HIS CD2 was actually ND1, HIS CE1 was NE2 and the nitrogens and oxygens of the ASN / GLN amide groups were actually the other way round. (Default is not to do this.) -X As -x, but only hydrogen bonds formed by HIS, GLN and ASN side-chains are included in the hydrogen bond list. This is a time-saving option when your purpose is to investigate the hydrogen bonding of HIS, GLN and ASN side-chains. [ The following options relate to the way the program is used in its "home" laboratory, and are only really included for completeness ] -i Do not attempt to load an sst file and input the secondary structure of the protein (default). -I Attempt to load an sst file and input the secondary structure. The program looks for a file with the filename "p????.sst" where ???? is the four-letter brookhaven code taken from the header line. -l Output in *.hb2 format (the default). -L Output in long *.hhb format, which is an extended version of the HBOND table in IDITIS (Oxford Molecular 1993), and includes the secondary structural information taken from an sst file. 2.4 Input Files HBPLUS.EXE requires a "clean" brookhaven (PDB) file, where all the atoms are accurately named and ordered, and no atoms have alternate locations. I expect that most "uncleaned" brookhaven files will work with HBPLUS, but if you want to be certain of your results, run a program to clean up the PDB file. HBPLUS will also attempt to find the an original "unclean" PDB file which contains the CONECT records, but will run the program anyway if that fails. If two files are named in the command line, then the second file is taken as being the old PDB file. If no such file is named, HBPLUS looks for a file with the name "p" + brookhaven code + ".pdb" in the current directory. If two files are named in the command line, then the first file is a "cleaned" file 2.5 Output File(s) Format The format of the main output file, the hydrogen bond list, is given in Table I below. ============================================================================ Table I: *.hb2 format 01-13 Donor Atom, including . . . 01 Chain ID (defaults to '-') 02-05 Residue Number 06 Insertion Code (defaults to '-') 07-09 Amino Acid Three Letter Code 10-13 Atom Type Four Letter Code 15-27 Acceptor Atom, same format as Donor atom 28-32 Donor - Acceptor distance, in Angstroms 34-35 Atom Categories - M(ain-chain), S(ide-chain) or H(etatm) - of D & A 37-39 Gap between donor and acceptor groups, in amino acids (-1 if not applicable) 41-45 Distance between the CA atoms of the donor and acceptor residues (-1 if one of the two atoms is in a hetatm) 47-51 Angle formed by the Donor and Acceptor at the hydrogen, in degrees. (-1 if the hydrogen is not defined) 53-57 Distance between the hydrogen and the Acceptor, in Angstroms (-1 if the hydrogen is not defined) 59-63 The smaller angle at the Acceptor formed by the hydrogen and an acceptor antecedent (-1 if the hydrogen, or the acceptor antecedent, is not defined) 65-69 The smaller angle at the Acceptor formed by the donor and an acceptor antecedent (-1 if not applicable) 71-75 Count of hydrogen bonds For example: HBPLUS Hydrogen Bond Calculator v 2.06 Jul 30 13:24:14 BST 1993 (c) I McDonald, D Naylor, D Jones and J Thornton 1993 All Rights Reserved. 1MBD <- Brookhaven Code "/idata/new/p1mbd.new" <- PDB file <---DONOR---> <-ACCEPTOR--> atom ^ c i cat <-CA-CA-> ^ H-A-AA ^ H- h n atom resd res DA || num DHA H-A angle D-A-AA Bond n s type num typ dist DA aas dist angle dist angle num -0002-LEU N -0153-HOH O 2.94 MH -1 -1.00 151.8 2.02 -1.0 -1.0 1 -0146-HOH O -0002-LEU O 2.78 HM -1 -1.00 -1.0 -1.00 -1.0 128.7 2 -0289-HOH O -0002-LEU O 3.45 HM -1 -1.00 -1.0 -1.00 -1.0 136.5 3 ============================================================================ In addition, the *.h file is used for the optional output file - the structure of the protein, with added hydrogens. The format is based on the Brookhaven PDB file. There is no temperature factor or occupancy in the ATOM or HETATM records. The only records included are HEADER, some REMARKs and the ATOM and HETATM records. HEADER PDB FORMAT FILE 30-JUL-93 1MBD REMARK 1 REFERENCE 1 REMARK 1 AUTH I.K.MCDONALD,D.NAYLOR,D.T.JONES,J.M.THORNTON ... ... REMARK 4 CONTACT I.K.MCDONALD AT ABOVE ADDRESS OR ON ELECTRONIC REMARK 4 MAIL AT \MCDONALD@UK.AC.UCL.BSM$ FOR INFORMATION ATOM 1 N VAL 1 -0.594 14.769 15.940 7.00 44.29 ATOM 2 1H VAL 1 -0.641 14.029 15.248 7.00 44.29 ATOM 3 2H VAL 1 -0.737 14.334 16.845 7.00 44.29 2.6 Adding New Residue Types If HBPLUS does not recognise an atom, or a residue, it will issue a warning statement. For instance "WARNING: Residue SO4 is not recognized by HBPLUS". If one of the unrecognised atoms has an role in hydrogen bonding - for instance it is a donor or acceptor, or connected to a donor or acceptor, you will probably want to define the residue or atoms within HBPLUS. This requires the five command line options -U/u (new residUe, with or without a similar old residue), -M (new atoM), -T (connecT) and -e/E (new donor/acceptor). You will probably find it wise to use the -f option and place the additional information in a separate file. The syntax for these commands is quite important. Residue names are always three characters long, and atom names are always four. If neccessary, use quotes to make the presence of trailing spaces obvious. Each bond in the list of bonds given to the -T command is in the form of two four-letter atom code with each bond terminated by a colon. For instance #HBPLUS option file to add SO4 residue -u SO4 #name the residue -M SO4 " S " #first atom -M SO4 " O1 O2 O3 O4 " #more atoms -T SO4 " S O1 : S O2 : S O3 : S O4 :" #the bonds -e SO4 " O1 " 2 #each oxygen can accept two H-bonds -e SO4 " O2 " 2 -e SO4 " O3 " 2 -e SO4 " O4 " 2 If the new residue is similar to one of the old residues and the atoms bear the same four-letter names, then the '-U' option can be used and the other atoms and covalent bonds added. For instance #HBPLUS option file to add NADP+ residue, called NAP -U NAP NAD #Similar structure and atom names to NAD -M NAP "AP2*AOP1AOP2AOP3" #the new phosphate group -T NAP "AP2*AOP1:AP2*AOP2:AP2*AOP3:" #Covalent bonds within the phosphate -T NAP "AO2*AP2*:" #Covalent bond to the phosphate -e NAP "AOP1" 2 #each oxygen can accept two H-bonds -e NAP "AOP2" 2 -e NAP "AOP3" 2 SECTION 3. THE SCIENCE ====================== 3.1 Introduction to Hydrogen Bonds In basic terms, a hydrogen bond (or H-bond) is an attractive interaction between two elctronegative atoms, a donor and an acceptor (Latimer and Rodebush 1920; Huggins 1971; Baker and Hubbard 1984; Ippolito, Alexander at al 1990; Stickle, Presta et al 1992). A hydrogen atom lies aligned between them and covalently bound to the donor. The donor attracts the electron on the hydrogen from its orbital towards the donor itself. This leaves a partial positive charge on the hydrogen, which is electrostatically attracted towards the elctronegative acceptor. The interaction is energetically favourable in a number of ways, including polarisation energy and covalent energy, but particularly the electrostatic energy. Some studies (eg Levitt and Perutz 1988) have suggested that the pi electron shells of aromatic rings may act as weak hydrogen bond acceptors. Because the pi electron shells are perpendicular to the plane of the aromatic ring rather than coplanar with it, the angles at the acceptor are formed by the perpendicular to the plane rather than the other covalent bonds (not including those to hydrogens) with the acceptor. 3.2 The Algorithm The algorithm for locating hydrogen bonds involves two steps. Firstly, finding the positions of the hydrogens, and secondly, calculating the hydrogen bonds. An interaction is counted as a hydrogen bond if (i) it is between a listed donor and acceptor (Table II) and (ii) the angles and distances formed by the atoms surrounding the hydrogen bond lie within the set criteria. If the donor and acceptor are only one or two covalent bonds apart, the interaction is not counted as a hydrogen bond. Cysteines are treated specially. Any two Cysteines which have their sulphur atoms within three Angstroms (this distance can be changed using the command line arguments) are defined as Cystines, and treated separately. In principle, Cystines can accept two bonds but cannot donate. ============================================================================== Table II - List of Hydrogen Bond Donors and Acceptors Donors 1. N (ie Main Chain NHs of recognised residues) 2. CYH SG, HIS NE2, HIS ND1, LYS NZ, ASN ND2, GLN NE2, ARG NE, ARG NH1, ARG NH2, SER OG, THR OG1, TYR OH, TRP NE1 3. Recognised donors of non-standard recognised molecules 4. Nitrogen atoms in unrecognised molecules 5. Oxygen atoms of recognised water molecules Acceptors 1. O (ie Main Chain COs of recognised amino - not imino - acid residues) 2. CYH SG, CSS SG, ASP OD1, ASP OD2, GLU OE1, GLU OE2, HIS ND1, MET SD, ASN OD1, GLN OE1, SER OG, THR OG1, TYR OH 3. Recognised acceptors of non-standard recognised molecules 4. Oxygen atoms in HETATM molecules (including waters) Atoms that may act as both donors and acceptors under the -X or -x options 1. HIS CD2, HIS CE1, ASN OD1, ASN ND2, GLN OE1, GLN NE2 Non-Standard Recognised Molecules 1. Standard Nucleotides C, A, U, G, T, also ATP. 2. Coenzymes COA, FMN, HEM, NAD 3. Small Molecules MTX, ACE, FOR 4. Amino Acids AIB, PHL, SEC, ALM, MPR, FRD, LYM, GLM, PPH, PGL, OLE, ABA, NLE, B2V, B2I, B1F, BNO, B2A, B2F, IVA, LOV, STA, PVL, CAL, PHA, DCI, AHS, CHS, MSE, ETA, PCA, ASX, GLX, UNK, CYH, CSS These can be listed with the -P option. ============================================================================== 3.2.1 Calculation of the Hydrogen Positions The programs makes one first pass through the protein structure calculating a locus for each donor heavy atom. The positions of the hydrogens are taken from Momany, McGuire et al (1975) and the main-chain NH hydrogen from Pauling and Corey et al (1951). They are illustrated in figures 2 and 3, together with information on planarity and loci. The precise bond angles and lengths are listed in table III. Each donor heavy atom in an amino acid is classified according to the hybridization of its electron orbitals and how many hydrogens or heavy atoms it is covalently bound to. The hybridization may be sp2 (trigonal planar) or sp3 (tetrahedral). The numbers of bound atoms are listed as 1, 2 or 3 hydrogens and then 1 or 2 DDs. The method of calculation is described below. The hydrogens bound to "sp2" and "sp3" hybridised atoms have different geometries. An atom with sp2 hybridisation has three orbitals projecting at about 120 degrees to each other, all in the same plane. These orbitals may or may not be part of covalent bonds to other atoms. For instance, ARG NE covalently bonds to two Cs and an H. In an optimal conformation, the C-N-C angle would be 120 degrees and the H would be exactly along the bisector. An atom with sp3 hybridisation has four orbitals pointing towards the corners of an imaginary tetrahedron. In an ideal conformation, the angles between any two orbitals would be 109.5 degrees. For instance, SER OG has sp3 hybridisation and a tetrahedral conformation. It is only attached to two atoms - a C and an H - and the C-O-H angle is still 109.5 degrees. 126.96.36.199 Hydrogens Bound to sp2 Hybridised (Trigonal Planar) Donors sp2 1H, 2DDs This includes NH groups on the main chain or Arg, His and Trp side chains. The donor atom is known to be attached to two DD heavy atoms and to the hydrogen. The angle DD1-D-DD2 is bisected by finding the mean of the directions of the vectors DD1-D and DD2-D. For main-chain groups the hydrogen is rotated within the plane of the peptide bond towards the CA in accordance with Pauling, Corey et al (1951). The hydrogen is placed a set distance away from D (usually 1.00 Angstroms for N donors) Fig 2: sp2 Hybridised (trigonal planar) hydrogen positions sp2 1H 2DDs sp2 1H 1DD sp2 2H 1DD DDD1 DDD2 DDD1 DDD2 DDD1 DDD2 H \ / \ / \ / | DD DD DD D | or | | /:\ D D D DD1 : DD2 \ / / \ : H H H H Planarity ? Y Planarity ? Y Planarity ? Y Fixed hydrogen ? Y Fixed hydrogen ? N Fixed hydrogens ? Y The locus is composed eg NH on main-chains of the two alternative eg ARG NH, ASN ND2, GLN OE2 Arg, His and Trp conformations shown side-chains above eg TYR OH sp2 1H, 1DD Although there are no standard amino acid donors that fall into this category, the Tyr OH, which is a combination of sp2 and sp3 hybridisation, behaves in a geometrically similar fashion and is modelled with this part of the algorithm. The H, DD, and one of the donor's lone electron pairs form a planar trigonal arrangement around D. the hydrogen may take one of the two positions where H, DD, DDD1 and DDD2 are coplanar and the H-D-DD angle takes the angle given in table 2. This locus is determined by first calculating hydrogen positions in a local co-ordinate system, and then transforming and translating them onto the donor atom. sp2 2H, 1DD This includes Asn and Gln amide groups and ARG NE. These all have a donor bound to three atoms that lie in the same plane and all angles at D are 120 degrees. DD, DDD1 and DDD2 also lie in the same plane. The two hydrogen positions are calculated in a local co-ordinate system before being transformed and translated onto the donor atom. 188.8.131.52 Hydrogens Bound to sp3 Hybridised (Tetrahedral) Donors Irrespective of the number of hydrogens, if there is only one DD heavy atom attached to the donor, then they may rotate around the D-DD bond, forming a circular locus. Steric hindrance is known to favour three particular staggered H-D-DD-DDD torsion angles 120 degrees apart. It is not obvious whether the expected locu of an sp3 hydrogen should be a circle or three alternative staggered points. The algorithm makes it is the former for single sp3 hydrogens and the latter for triple sp3 hydrogens. sp3 1 H, 1 DD These include OH on Ser and Thr, and SH on Cyh. The centre of the circular locus is found by projecting the D-DD bond a distance that depends on the lengths and angles for the group in question. A default position, staggered relative to the D-DD line, is calculated in a local co-ordinate system, then transformed and translated. the circular locus that the hydrogen is allowed to move along is normal to the D-DD line. sp3 3 Hs, 1 DD The only example of this is LYS NZ, and terminal amino groups. the combined locus is made up of three alternative points, equally spaced and staggred relative to DDD, where DDD is one covalent bond beyond DD from D. The positions are calculated using a local co-ordinate system and relevant bond lengths and angles, and transformed and translated onto the donor. Fig 3: sp3 hybridised ( tetrahedral orientations ) hydrogen positions sp3 1H 1DD sp3 3Hs 1DD H -> (goes in a circle) *H H H Planarity ? N \ \|/ (although the H marked * is D D in the same plane as D, DD | | and DDD) DD DD \ \ Fixed Hydrogens ? Y DDD DDD Planarity ? N H Fixed Hydrogen ? N / *H-D -DDD <- DD is hidden by D The hydrogen may swivel round \ All three DD-D-H angles are the DD-D axis towards the H equal. acceptor. eg SER OG, THR OG1, CYH OG1 eg LYS NZ, terminyl amino groups =================================================================== TABLE III - DONOR GROUP GEOMETRIES FOR CALCULATED HYDROGENS Name of Donor Atom H Bonds Angles etc D-H ==================================================================== HIS ND1, HIS NE2 sp2 1H 2DD DD1-D-H = DD2-D-H 1.00 ARG NE , TRP NE1 -------------------------------------------------------------------- TYR OH sp2 1H 1DD DD-D-H = 110, 250 1.00 DD1,DD2,D,H are planar -------------------------------------------------------------------- ASN ND2, GLN NE2 sp2 2H 1DD DD-D-H = 120 1.00 ARG NH1, ARG NH2 DDD-DD-D-H = 0, 180 ==================================================================== CYH SG , CYS SG sp3 1H 1DD DD-D-H = 96 1.33 -------------------------------------------------------------------- SER OG , THR OG sp3 1H 1DD DD-D-H = 110 1.00 -------------------------------------------------------------------- LYS NZ, any amino sp3 3H 1DD DD-D-H = 110 1.01 terminus DDD-DD-D0H = 180 ==================================================================== Backbone N sp2 1H 2DD (C-N-H)-(CA-N-H) = 4 1.00 C, CA, N, H are planar ==================================================================== H hybridisation - sp2 (trigonal planar) or sp3 (tetrahedral) Bonds number of covalently attached hydrogens (H) and heavy atoms (DDs) Angles Angles and conditions used to precisely define hydrogen position D-H D-H distance for the calculated hydrogen in Angstroms ==================================================================== 3.3 Selecting Potential Hydrogen Bonds Once the hydrogen positions have been determined as far as possible, each donor/acceptor pair is examined in turn to see if it fits the geometric criteria. It is intended that geometric criteria will tend to be determined by the purpose of the study. For maximum comparability, this program defaults to the same minimum angles and maximum distances as Baker and Hubbard (1984). These are : - Maximum Distances D-A of 3.9 Angstroms H-A of 2.5 Angstroms Minimum Angles D-H-A of 90.0 degrees D-A-AA of 90.0 degrees H-A-AA of 90.0 degrees Maximum Angles D-A-AX of 20.0 degrees } for amino-aromatic interactions H-A-AX of 20.0 degrees } (AX is at L to aromatic plane) The -d -h -b -B -a and -A command line options exist to allow the criteria to be customised. There are rules to cover contingencies. If the hydrogen on the donor is not fixed then the point on the hydrogen's locus that is closest to the acceptor being investigated is selected. If no position was given for the hydrogen on the donor (for instance, for a water oxygen or an unrecognised nitrogen) then it is assumed to be directly between the donor and acceptor, one angstrom away from the donor. If the acceptor is covalently bound to more than one heavy atom, yielding more than one possible "angle at the acceptor", the lower value is given. The algorithm is slightly inflexible. It finds potential hydrogen bonds rather than real ones, and frequently, such as in the case of those donors for which the hydrogen could not be positioned (eg serine, threonine or tyrosine oxgygens), the hydrogen bonds can be mutually exclusive. If a pair of atoms could act both as donor and acceptor to each other, for instance a "SER OG " and a "HOH O ", then they are listed as forming two hydrogen bonds. If more than one location is given for any particular atom then the different locations are treated as different atoms that simply happen to have the same name. In these circumstances, a donor / acceptor pair can have two hydrogen bonds listed with different geometries. 3.4 Orienting ASN, GLN and HIS side-chains 3.4.1 Introduction To define a structure by X-ray crystallography a protein must be modelled into an electron density map that at usual resolutions rarely shows hydrogen atoms and shows little or no difference between carbon, nitrogen and oxygen atoms. There are now a few structures (three in the October 1993 release of the Brookhaven Protein Database (Bernsteine et. al. 1977)) at resolutions as high as 1.0A where the carbon, nitrogen and oxygen atoms can sometimes be differentiated and some of the hydrogens can be observed. However, given the diffracting power of most protein crystals, these will probably remain the exceptions. For the majority of side-chains the atoms can be uniquely identified from the shape of the electron density map, but for asparagine, glutamine and histidine, whose side-chains appear symmetrical in the electron density, some specific atoms can only be identified on the basis of their environment, principally their hydrogen bonds. It is also difficult to differentiate between the three different protonation states of histidine. As the imidazole ring of histidine has a pK (6.5-7.0) close to physiological pH (~7-8) (Matuszak and Matuszak, 1976), both the basic and charged forms occur in vivo. The positively charged form is protonated on both imidazole nitrogens, whilst the basic form is protonated on only one imidazole nitrogen, and occurs as two tautomers which differ in which nitrogen is protonated. NMR studies on His at basic pH and in the polypeptide antibiotic Bacitracin suggested that the basic His is more usually protonated on NE2 rather than the ND1 (Reynolds et al 1973). Because both charged and basic forms of histidine are stable, histidine often participates in catalysis, and is found in the active sites of enzymes (e.g. serine proteases such as chymotrypsin) or as an axial ligand in metalloproteins such as the cytochromes (e.g. Cytochrome b5). In chymotrypsin, for instance, the active site histidine is involved in every step of catalysis and changes protonation state four times in the entire catalytic cycle. The chemistry of Asn and Gln is simpler. Here the problem is only to distinguish between the side-chain nitrogen and oxygen atoms. Distinguishing between the two, if the hydrogens are not visible, can become difficult however because some nearby side-chain atoms or water molecules can act as either donors or acceptors. Since the nitrogen can donate two H-bonds and the oxygen accept two H-bonds, it is sometimes possible to use the information on whether they form one or two hydrogen bonds to differentiate between the alternative conformations. 3.4.2 The Algorithm The study used the list of hydrogen bonds, including those that could only occur if the Asn, Gln and His side-chains were assumed to be in the alternative conformations. This algorithm works on the assumptions that (i) if an atom is accessible to solvent, however slightly, it can form a hydrogen bond to solvent and (ii) hydrogen bonds that are visible in X-ray structures are generally more energetically favourable than those implied by accessibility to solvent. Assumption (ii) is justified because if any atom appears in the electron density its location is well defined, and it is therefore tightly bound. If the H-bonded water molecule is not visible then by implication the binding site is not as well defined and the H-bonds are weaker. It is generally accepted that, of atoms which can donate more than one or accept more than one hydrogen bond, the additional hydrogen bonds are not as energetically favourable as the first hydrogen bond. Since nearly as many Asn and Gln side-chain donors and acceptors form two visible hydrogen bonds as form one, this implies a significant but lesser energetic gain. Therefore, when analysing Asn and Gln side-chains, whether atoms formed one hydrogen bond rather than two, was used as a "tie-breaker" in cases where the two alternative conformations had the same numbers of both buried unsatisfied atoms and of atoms satisfied by implied H-bonds to solvent. Both hydrogen bonding atoms were examined for both conformations of each Asn, Gln and His side-chain, and classed as either satisfied by a visible hydrogen bond ("satisfied"), satisfied by an "implied" hydrogen bond to solvent ("implied"), or unsatisfied by either visible or implied hydrogen bonds ("unsatisfied"). In His residues, the H-bonds formed by (i) ND1 and NE2 and (ii) CD2 and CE1 were examined. We would expect the atoms labelled as nitrogen to be involved in H-bonds rather than the carbons. Occasionally we found that both nitrogens accepted H-bonds, and neither donated. In principle, since it is not possible for both nitrogens to accept H-bonds, only one of the atoms is counted as satisfied in this situation. In the case of Asn (Gln) residues this means examining the OD1(OE1) and ND2(NE2) twice - once including H-bonds donated by the ND2(NE2) and accepted by the OD1(OE1), and once vice-versa. The degree of hydrogen bond satisfaction of either conformation of any Asn, Gln or His side- chain is described by giving a pair of classifications, one for each atom. For instance "unsatisfied and satisfied", or "implied and implied". The degrees of satisfaction can be compared between the PDB and the alternative conformation. The side-chain is classed as follows: Highly Optimal, if there is an "unsatisfied" atom in the alternative conformation but not in the PDB conformation, or if there are two "unsatisfied" atoms in the alternative conformation but only one in the PDB conformation. Slightly Optimal, if the hydrogen bonding potential is more highly satisfied in the PDB orientation than in the alternative, but the hydrogen bonding patterns does not qualify as "Highly Optimal". For instance, if the PDB conformation is "Satisfied and Satisfied" but the alternative conformation is "Implied and Satisfied". Indifferent, if the PDB and the alternative conformation are equally favourable or unfavourable. Slightly Suspect, if the alternative conformation is more favourable than the PDB conformation, but the number of buried unsatisfied atoms is the same for both conformers (i.e. the converse of "slightly optimal"). Highly Suspect, if the number of buried unsatisfied atoms is lower for the alternative conformation (i.e. the converse of "highly optimal"). 3.4.3 Accessibility and Implied Hydrogen Bonds A buried atom is defined as one having a zero solvent accessibility according to an implementation of the Lee and Richards (1971) algorithm calculated by the program ACCESS (Hubbard, 1992, 1994) with a probe size of 1.4A. Any hydrogen bond donor or acceptor with non-zero accessibility to solvent and no visible hydrogen bonds, is regarded as forming an implied hydrogen bond with solvent. SECTION 4. REFERENCES ===================== Baker, E. N. and Hubbard, R. E. (1984). "Hydrogen Bonding in Globular Proteins." Prog Biophys Molec Biol 44: 97. Bernstein, F. C., Koetzle, T. F., et al. (1977). "The Protein Data Bank: A computer based archival file for macromolecular structures." J Mol Biol 112: 535. Gardner, S. and Thornton, J. M. (1992). IDITIS. Oxford, Oxford Molecular Limited. Hubbard, S. (1992). "ACCESS", University College London. Hubbard, S. (1994). "NACCESS", Heidelberg. Huggins, M. L. (1971). "50 Years of Hydrogen Bonding Theory." Angewandte Chemie International Edition 10: 147. Ippolito, J. A., Alexander, R. S., et al. (1990). "Hydrogen Bond Stereochemistry in Protein Structure and Function." J Mol Biol 215: 457. Latimer, W. M. and Rodebush, W. H. (1920). "Polarity and Ionization." J Am Chem Soc 42: 1419. Lee, B. and Richards, F. (1971). J Mol Biol, 55: 379-400. Levitt, M. and Perutz, M.F. (1988). J Mol Biol 201: 751-754. Matuszak, C.A. and Matuszak, A.J. (1976) J Chem Educ 53: 280-284 McDonald, I.K. and Thornton, J.M. (1994). J Mol Biol 238: 777-793 Mitchell, J. B. O. (1990). "Theoretical Studies of Hydrogen Bonding" PhD Thesis, Churchill College Cambridge Momany, F. A., McGuire, R. F., et al. (1975). "Energy Parameters in Polypeptides. VII. Geometric Parameters, Partial Atomic Charges, Nonbonded Interactions, Hydrogen Bond Interactions, and Intrinsic Torsional Potentials for the Naturally Occuring Amino Acids." J Phys Chem 79(22): 2373. Pauling, L., Corey, R.B. and Branson, H.R. (1951). "The Structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain" P Nat Acad Sci USA 37: 205-211. Reynolds, W.F., Peat, I.R., Freedman, M.H. and Lyerla, J.R., Jr. (1973) J Amer. Chem Soc, 95: 328-331. Stickle, D. F., Presta, L. G., et al. (1992). "Hydrogen Bonding in Globular Proteins." J Mol Biol 226: 1143. Vriend, G., Berendsen, H., et al. (1991). "Stabilization of the neutral protease of Bacillus Stearothermophilus by Removal of a Buried Water Molecule." PE 4(8): 941.