Contents

• Introduction
• Example
• Syntax
• Required Software

Introduction

Using the -autobondrot command line flag, Probe can generate multiple molecular conformations by rotating atoms around defined dihedral axes and perform other transformations. To use this function you must construct an input script defining rotatable bonds, etc. Usually called .rotscr files, they are quite similar to the @bondrot sections in a kinemage file. The probe command iterates over each conformation and generates a tabular output of the probe score and the conformation angles.

Atomic coordinates for specific conformations (in PDB format) can be output.

A simple example of how to use -autobondrot is to explore the range of probe scores for all conformations of a given amino acid sidechain to analyze how it interacts with itself and its static neighbors.

Example - How to make a sidechain conformation map in unix or linux

These example files are available packaged with the autobondrot scripts from our web site.

First, take the PDB file (with hydrogens from Reduce) and use Prekin to make a rotatable group or mutation. In this example, tyrosine 61 in the PDB file 1ah7H is examined and we used Prekin to create 1ah7y61bondrot.kin.

The script for sampling the scores is created with mkrotscr, which translates the @bondrot sections of the .kin file into the proper syntax for the .rotscr file.

When running mkrotscr, the residue and PDB file are listed after the kinemage filename, so that mkrotscr can construct a plausible suggested command line for a sidechain rotation in the .rotscr file. Executing this command will invoke Probe to calculate contacts and summarize the result as a probe score. The proposed Probe command reads the autobondrot information until the END_OF_INPUT marker is seen. To make this file an executable UNIX shell script simply execute:

When the residue numbers are unique, an alternative way to create this rotation script is to use pdb2rotscr, a simple command script which combines Prekin and mkrotscr. In our example, the command would be as follows:

This .rotscr file needed to be edited because Mage wrote out a third rotatable bond (the OH) which we did not want to explore. In addition, we modified the bounds of the search of chi2 from 0-359° (by 5) to 0-179° (by 10) because the phenyl ring is symmetric and it is not necessary to scan chi2 as finely as chi1. To make the file more informative, we changed the rotation names from "rot1" and "rot2" to "chi1" and "chi2". We only want a torsional penalty applied to chi1 so we took out all but the first "cos" record.

A duplicate copy of the atom record for C-zeta was deleted for neatness. Finally, since this residue does not have any branch points which require independent nested rotations we can safely delete the SAVE/RESTORE pair: ( and ). The final result is as follows:
probe -q -stdbonds -3 -drop -once "file1" "file1 | file2 alta not water not(sc 61)" -auto - 1ah7H <<END_OF_INPUT

atom      1  cb  tyr    61      34.219  17.937   4.659  1.00  0.00
bondrot:chi1:78.7:  0:359:5:33.138:18.517: 5.531:34.219:17.937: 4.659
cos:-3:60:3:
atom      1 1hb  tyr    61      34.766  18.777   4.206  1.00  0.00
atom      1 2hb  tyr    61      34.927  17.409   5.315  1.00  0.00
atom      1  cg  tyr    61      33.836  16.989   3.546  1.00  0.00
bondrot:chi2:-11.8:  0:179:10:34.219:17.937: 4.659:33.836:16.989: 3.546
atom      1  cd1 tyr    61      32.578  16.433   3.418  1.00  0.00
atom      1  cd2 tyr    61      34.803  16.657   2.603  1.00  0.00
atom      1  ce1 tyr    61      32.294  15.554   2.393  1.00  0.00
atom      1  ce2 tyr    61      34.520  15.798   1.551  1.00  0.00
atom      1  cz  tyr    61      33.249  15.259   1.456  1.00  0.00
atom      1  hd1 tyr    61      31.793  16.694   4.142  1.00  0.00
atom      1  hd2 tyr    61      35.813  17.084   2.693  1.00  0.00
atom      1  he1 tyr    61      31.299  15.089   2.328  1.00  0.00
atom      1  he2 tyr    61      35.291  15.550   0.807  1.00  0.00
atom      1  oh  tyr    61      32.991  14.372   0.421  1.00  0.00
atom      1  hh  tyr    61      33.803  14.287  -0.156  1.00  0.00
END_OF_INPUT

We make the script executable with:

A table of scores is generated by running this rotation script, which feeds the records up to the END_OF_INPUT to Probe after the -auto flag. This system makes use of the <<LABEL ... LABEL UNIX syntax for inline data. The run may take several minutes.

Finally, the scores can be contoured by Kin2Dcont and viewed in Mage. The range of contour levels can be customized as required.

A similar program, Kin3Dcont, makes contours of 3 dimensional datasets. Our data is uniformly sampled (-sampled) and the data are cyclic so that 360 deg is the same as 0 deg (-wrap # # # #). This is why we only sampled the data up to 355 and 175 degrees in the two dimensions. The -gxy 5 10 and -sxy 4 8 control the size of the sampling grid in each dimension (5° by 10°) and the size of the spot filter (sdev x = 4°, sdev y = 8°) used to smooth the data. If the grid spacing is the same in each dimension a combined setting may be used (-g# and -s#). The spot filter can be varied to either increase smoothing (-sxy 10 20) or eliminate any smoothing (-s0). For sampled data, setting the spot size about equal to the grid spacing seems to work well.

The Elements Of An Autobondrot Script

The input script for autobondrot is composed of records of the following types:

The information on each record (except ATOM) is separated by colons and must be all on one line. Any line beginning with a # is ignored, providing a convenient means of including comments in the script. For rotations, BONDROT and COS records head each section of ATOM records which are subject to the same rotation.

*BONDROT - Both the current angle of the rotatable bond and the range of angles to be sampled are defined on the BONDROT record, along with the end points of the axis. It consists of eleven data fields: the axis name, current angle, beginning rotation angle, final rotation angle, the amount of rotation, and finally the x, y and z values of the beginning and end of the rotation axis.

The axis name does not have to be a number (e.g., chi1 or phi).

The current angle can be measured with the measures tool within Mage.

Any atoms listed before the first BONDROT will be output but their coordinates will not be altered. Subsequent BONDROT records are treated as nested rotations. Use SAVE and RESTORE records to control where these nested rotations begin and end.

The nested chi1, chi2 rotations in our tyrosine example are:

   bondrot:chi1: 78.7:  0:359:5:   33.138:18.517:5.531:  34.219:17.937:4.659
      ... atoms rotate about chi1 ...
   bondrot:chi2:-11.8:  0:179:5:  34.219:17.937:4.659:  33.836:16.989:3.546
      ... atoms rotate about chi1 then chi2 ...

Here the chi1 dihedral axis is defined by the C-alpha and C-beta atoms while chi2 is defined by C-beta and C-gamma.

Note that the axis does not have to be along a bond. For example, an entire molecule could be rotated around the coordinate axes.

*TRANS - Probe can also translate atoms. In this case, the axis defines the direction of the translation, and instead of angles we have angstroms.

*NULL - A null transformation does not modify the position of any atoms. It has no data fields.

*COS - after the transformation record (e.g., BONDROT) one or more records can be provided which define a bias function. The most generally useful is the COS record which is used to add a torsional penalty to Probe scores as we rotate around a dihedral. It consists of up to four data fields: scalefactor, phase offset, frequency, and a seldom used offset which defaults to 1. In our example, we use a torsion only with chi1:

This describes the following function: -3*(1 - cos(3*(x - 60)))/2, a cosine with three peaks with a value of 0.0 at -60, +60 and 180 and a value of -3.0 at 0, 120 and 240.

More complicated functions may be built frome those provided by ganging-up more than one COS, POLY or CONST record.

*POLY - A polynomial can be built from one or more POLY records. It has three data fields: a scalefactor, offset and degree.

results in the following quadratic: 5.0*(x - 0.0)^2

*CONST - a constant value can be added to the score. This record has one data field: the value.

*ATOM - Rotations and other transformations operate on ATOM records, listing each atom which is subject to the bond rotation. ATOM records may also be supplied prior to any BONDROT record, in which case they are not subject to any rotation. All ATOM records are in PDB-atom record format.

The critical data fields are the full atom name, residue name, residue number and x,y,z position. The format requires data to be in specific columns.

   atom        1hb  tyr    61      34.766  18.777   4.206  1.00  0.00

*SAVE - Abbreviated "(", SAVE saves the current transformation on a stack.

*RESTORE - Abbreviated ")", RESTORE backs up to the last previously saved transformation. Save and restore are used to organize transformations for branching groups. For example when rotating the all sidechain angles of an isoleucine (including the methyl groups) the following SAVE/RESTORE grouping is required:

   bondrot:chi1: ...
   ...
   (
   bondrot:chi2: ...
   ...
   bondrot:CD1 meth: ...
   ...
   )
   bondrot:CG2 meth: ...
   ...

*GO - GO records are optional. If included, they consist of a set of angles, one for each BONDROT or TRANS, in the same order. Many GO records can be included in a single .rotscr file.

If there are no GO records, autobondrot will generate all permutations of conformations defined on the BONDROT records. If one or more GO records are found, autobondrot will not grind through all these permutations but will instead run the command on the specific conformation listed. A series of GO records will sample a discrete set of conformations. This statement sets the chi1 and chi2 limits of 60° and 90°, respectively.

If the -verbose option (versus -quiet or -q) is used, Probe will write atom records for the transformed coordinates to standard error, once for each GO record. This may be useful as a way of generating coordinates for a specific orientation. To capture this standard error output in a file along with standard output, UNIX provides the command line syntax command >& outputfile

*Include files - These records are also optional. If a line begins with an @ sign the following text is treated as a filename (@filename) and Probe attempts to start reading autobondrot commands from this file, to the end, before continuing on with the current file. Include files can be nested. The most common use for includes is to add in pre-defined batches of go statements which sample set regions of conformation space for a residue type.

Required Software

An ensemble of programs, available for UNIX or LINUX, is required to run Probe with the -autobondrot flag.

Each of these programs must be placed in a directory listed in your PATH. If the download process has not made the files executable, each must be made executable with the command:

Awk is an interpreted scripting language for processing text files which is available on almost all UNIX systems.

Probe is used in the example above to calculate contact dot scores.

Reduce is listed above because hydrogens are necessary for successful use of Probe. Add the hydrogens to the PDB file before running prekin and the rest of the operations listed above.

See Word et al. (2000) "Exploring steric contraints on protein mutations using MAGE/PROBE", Protein Sci. 9, 2251-2259 for an application example.

e-mail:
J. Michael Word or
David C. Richardson
URL: http://kinemage.biochem.duke.edu
Biochemistry Department
Duke University
Durham, NC USA 27710