X-PLOR - Example Scripts

X-PLOR

3 Example Scripts

Files in the X-PLOR release tree are contained in nmrlib, test, toppar, tutorial, xtallib and xtalmacro directories.

The nmrlib and xtallib directories contain standard library data and should not normally be changed.

The toppar directory contains standard parameter and topology files and will not normally be altered.

The macro directory contains a library of macros written in the X-PLOR scripting language. These macros may be called by the input scripts.

The test directory contains a set of input scripts that can be used to quickly test the proper operation of the X-PLOR program and help diagnose any problems that may occur.

This release of the X-PLOR software contains numerous example scripts that you can use as templates for your calculations with the X-PLOR program. The scripts are located in a set of subdirectories within the tutorial directory, described in more detail in the rest of this chapter. Some comments describing the purpose of each script and the method of implementation using the X-PLOR scripting language are provided within the scripts themselves.

Running X-PLOR

Input scripts may be used to run the X-PLOR program directly from the UNIX command line, with output from the calculation directed to a log file. For example:


xplor.exe <script.inp> script.log

Scripts for many X-PLOR applications may also be generated, executed, and the results analyzed with the graphical user interfaces in the Insight II modules Xsight or NMR_Refine and in the QUANTA program.

General example scripts

X-ray crystallography scripts

dataconversion/

This directory contains an example script, data_convert.inp, for converting a file containing a set of reflection data into the X-PLOR format. The conversion is carried out using the utility program xplor_data_convert, provided with the X-PLOR distribution. See Appendix B for a description of the format and a description of how to use this utility program.

mad/

The example scripts in this subdirectory provide the capability of carrying out probabilistic MAD phasing with the X-PLOR program.

The mad_merge.inp script merges reflection data from several files (with each file corresponding to a data set collected at a different wavelength) into one file. Once merged, the mad_scale.inp script computes scale factors for each data set relative to the reference data set and places the data on an approximately absolute scale.

The mad_analyse.inp script computes dispersive and anomalous diffraction ratios and the mad_histogram.inp script computes histograms of the dispersive and anomalous differences.

An example of how to carry out heavy atom refinement against the MAD data and the computation of phase probability distributions is given by the mad_refine.inp script.

Scripts are also available for the several types of map calculation that might be needed for a MAD structure determination. The mad_map.inp script computes fom-weighted electron density maps from MAD phases. The mad_diff_map.inp script computes fom-weighted dispersive difference Fourier maps between wavelengths and the mad_diff_as_map.inp script computes fom-weighted anomalous difference Fourier maps at a wavelength.

For more information on MAD phasing with the X-PLOR program see Chapter 2, Methodology.

pdb_xtal_submission/

This directory contains an example script, xtal_submit.inp, which automatically sets up much of the information required by the Protein Data Bank when a protein structure is deposited.

xtal_analysis/

The four scripts within this subdirectory provide tools for analyzing crystallographic data and for estimating co-ordinates errors from this data.

The i_histogram.inp script is used to generate a histogram of structure factor intensities.

The wilson.inp script is used to compute a Wilson plot and provides a quasi-absolutes scale and overall B-factor.

The luzzati.inp script provides a cross-validated Luzzati coordinate error estimate and the sigmaa.inp provides an estimate of the co-ordinates errors based on a cross-validated _A calculation.

xtal_free_r/

The setup_free_r.inp script can be used to select the random set of reflections that will be used for cross-validating macromolecular structure refinements.

The extend_free_r.inp script can be used to retain and expand the test reflection data into another, higher resolution, data set.

xtal_maps/

This subdirectory contains four example scripts, illustrating the calculation of various types of electron density map.

The nfo-mfc_phicalc_map.inp script shows how to calculate an (nFo-mFc, phicalc) map with cross-validated _A weighting.

The sa_omit_map.inp script shows how to calculate an annealed omit map with cross-validated _A weighting.

The fo-fo_phicalc_map.inp script shows how to calculate an (Fo-Fc, phicalc) map with cross-validated _A weighting

The fo_phiobs_map.inp script shows how to calculate a (Fo, phiobs) map with fom-weighting

xtal_torsion/

The two scripts, torsion_constant.inp and torsion_slow.inp, provide examples of a constant temperature torsion angle dynamics protocol and slow-cooling torsion angle dynamics protocol respectively. A new script, torsion_slow_ml.inp, exemplifies a minor adaptation of these scripts for optimal use with the maximum-likelihood targets. The torsion angle dynamics protocols have a greater radius of convergence than conventional simulated annealing and would be used to refine a relatively inaccurate model.

xtal_waterpick/

The waterpick.inp script calculates an electron density map and picks ordered water molecules at the peak densities near the protein surface.

xtalmr/

The scripts within this subdirectory are for molecular replacement calculations (Patterson searches and refinement). The usage and sequence in which these scripts are run depends on the type of molecular replacement problem that is to be solved.

1. Simple case of a rotation and translation search for a single molecule

1. Run the self_rf.inp script to carry out a self-rotation function to check that there is no non-crystallographic symmetry.

2. Run the rotation.inp script or the rotation_direct.inp script to determine the orientation of the search model in the crystal.

3. Run the tftranscross.inp script to solve the translation function for the oriented search model within the crystal.

2. Simple case of a rotation and translation search for multiple molecules in the crystal

1. Run the self_rf.inp script to carry out the self-rotation function to determine the non-crystallographic symmetry.

2. Run the rotation.inp script or the rotation_direct.inp script to determine the orientation of the first copy of the search model in the crystal.

3. Run the tfcrosstrans.inp script for the translation function search for the first molecule.

4. Run the tfcrosstrans2.inp script for the translation function search for the second molecule with the first molecule placed and fixed. (Note: The orientation of the second molecule is taken from the rotation function list or is known from the NCS symmetry.)

3. Complex case consisting of single or multiple molecules containing single or multiple domains

1. Run the self_rf.inp script to carry out the self-rotation function to determine the non-crystallographic symmetry.

2. Run the rotation.inp script or the rotation_direct.inp script to determine the orientation of the search model in the crystal.

3. Run the searchv.inp script to carry out a local rotation function search of the first domain of the molecule.

4. Run the searchc.inp script to carry out a local rotation function search of the second domain of the molecule.

5. Run the tfcross.inp script for the calculation of a relative PC translation search between both domains.

6. Run the tfcrosstrans.inp script to carry out the translation search for the first molecule.

7. Run the tfcrosstrans2.inp script to carry out a translation search for the second molecule with the first molecule placed and fixed.

4. Complex case with single or multiple molecules with single or multiple domains using PC refinement

1. Run the self_rf.inp script to carry out the self-rotation function to determine non-crystallographic symmetry.

2. Run the rotation.inp script or the rotation_direct.inp script to determine the orientation of the search model in the crystal.

3. Run the filter.inp script to carry out PC refinement on the rotation function solutions.

4. Run the translation1.inp script to carry out a translation search on the first molecule.

5. Run the translation2.inp script to carry out a translation search on the second molecule.

6. Run the translation12.inp script to obtain the relative translations between the first and second molecule.

7. Run the rigid.inp script for rigid body refinement on the oriented and positioned molecules.

Other example scripts in this subdirectory are packing.inp, which calculates a packing function, phased_translation.inp, which calculates a phased translation function, unitcell.inp which is an example showing the explicit generation of molecules related by crystallographic symmetry and elbow.inp, which shows how to modify an elbow angle in an Fab fragment.

xtalrefine/

This subdirectory contains example scripts for macromolecular structure refinement. Before running these scripts it is necessary to generate the protein structure file for your molecule (use scripts in the generate/ subdirectory). You should normally also set up a test set of reflection data to cross-validate the refinement (use scripts in the xtal_free_r/ subdirectory). In particular, note that for the maximum-likelihood refinement targets, which are usually preferred and are the current default, a test set of reflection data is required.

The following are the basic steps that are needed to refine an atomic model.

1. Run the check.inp script to calculate the weight (wa) that scales the gradient in the X-ray energy to the gradient in the chemical energy for the target function.

2. If the model geometry is poor, run the prepstage.inp script to correct these errors.

3. Run the positional.inp script for a conventional minimization or the slowcool.inp script for a simulated annealing refinement. Alternatively, for structures with larger errors, you may wish to run the torsion angle dynamics scripts in the xtal_torsion/ subdirectory. If multiple refinement trials are carried out (that is, refinements beginning with different random velocity assignments) pick the structure with the lowest free R value for further refinement. It is recommended to carry out at least two trials. However, it is much better to carry out five to ten trials if sufficient computing resources are available.

To refine the atomic thermal parameters the brefinement.inp script, which performs restrained refinement of individual isotropic B-factors, or the bgroup.inp script, which performs group refinements of main and side chain atoms on a residue by residue basis, may be used. The minor adaptations needed to use the maximum likelihood target are exemplified by the script, brefinement_ml.inp.

Other scripts that may be useful are ncs_strict.inp and ncs_restrain.inp, which show how to set up non-crystallographic symmetry information. The alternate.inp script shows how to set up restraints for discrete disorder in the X-PLOR program and the anomalous.inp script shows how to incorporate the effects of anomalous scattering into the refinement.

The setup_bulksol.inp script is designed to set up a bulk solvent model, which may allow incorporation of lower resolution data into the refinement (see slowcool_with_bulk.inp for an example). If you use this script you should check that the solvent parameters (the solvent density and B-value) are reasonable and that there is a significant drop in R value, especially at low resolution. If the results are unsatisfactory, try fixing either the solvent B-factor or the solvent density level. The reflection file that is written out should then be used in all subsequent jobs (including map calculations). Note that the same lower resolution bound should be used in all subsequent jobs.

The baoverall.inp script may be used to apply an overall anisotropic B-factor refinement. Note that this correction modifies the FOBS data and the reflection file containing this modified data that is written is then used in subsequent refinement jobs.

A script is also available to set up a resolution-dependent weighting scheme of the type used in ProLSQ (resolution_dependent.inp) and an example of how to use this weighting scheme in a refinement is provided (slowcool_resdep.inp). However, the maximum-likelihood targets available in this version of X-PLOR generally supercede the use of this weighting scheme.

NMR structure determination scripts

aria/

Example files for the use of the ARIA methodology are provided in four subdirectories (analysis, assign, data, protocol and toppar). Two files, initialize.xplor and project.xplor illustrate overall control of the application of this methodology. More information on using ARIA is found in Chapter 2, Methodology.

nmr/

This directory contains a set of scripts for NMR structure determination by distance geometry and simulated annealing. Important new scripts are sa_tad_stein.inp and sa_tad_all.inp, which are protocols for structure determination using torsion angle dynamics.

nmr_ensemble/

This directory contains scripts for multiconformer refinement. The *_cv_* scripts perform complete cross-validation as a function of the number of conformers to find out which number of conformers best fits the NMR data.

When using these scripts it should be noted that, since no ensemble averaging is yet available for J-coupling and dihedral angle restraints, these restraints should be duplicated for each conformer in the ensemble (that is, use specific SEGIDs to define dihedral angle restraints for each conformer in the ensemble). It should also be noted that hydrogen bond restraints should not be ensemble averaged. Therefore, as with dihedral angle restraints, duplicate the hydrogen bond restraints for each conformer in the ensemble using specific SEGIDs.

Four example scripts are available for each of three different cases.

1. Monomeric protein with the entire structure treated as a multi-conformer

The script ensemble_cv.inp carries out complete cross-validation as a function of the number of conformers in the ensemble.

The script ensemble.inp may be used to carry out the multiconformer refinement. This script should be used with the best number of conformations found using the ensemble_cv.inp script.

The script rmsd_ens.inp generates single conformer PDB files from multiconformer files and calculates an average structure.

The script pmrefine_ens.inp generates an average representation of the ensemble of conformers following a probability map refinement. The script uses the structures generated using ensemble.inp and rmsd_ens.inp.

2. Dimeric protein with the entire structure treated as a multiconformer

Scripts for this case are analogous to the examples for the monomeric protein. The names of the four scripts are ensemble_dimer_cv.inp, ensemble_dimer.inp, rmsd_ens_dimer.inp and pmrefine_ens_dimer.inp.

3. Monomeric protein in which only a loop region is treated as a multiconformer

Scripts for this case are analogous to the examples for the monomeric protein. The names of the four scripts are ensemble_mloop_cv.inp, ensemble_mloop.inp, rmsd_ens_mloop.inp and pmrefine_ens_mloop.inp.

nmr_relaxation/

The directory contains five example scripts for relaxation matrix refinement.

The script, spectrum.inp, is an example of prediction of the spectrum from a 3D structure. The script, refine.inp, illustrates refinement against NOESY intensities. The example script, multiplesolvent.inp, shows how to set up simultaneous refinement with H₂0 and D₂0 spectra. The script, rfactor.inp, illustrates how to calculate different R values for various restraint classes and intensity shells. The script, taugrid.inp, shows how to set up a grid search for the correlation time that produces the lowest R value.

See chapter 21 of the X-PLOR 3.1 manual for a detailed description of these scripts. Also, see Chapter 2, Methodology, in this manual for information on very fast direct NOE refinement.

nmr_shift_coup/

This directory contains four example scripts for ¹H and ¹³C chemical shift and J-coupling refinement.

The script mini_shift_coup.inp is an example of refinement using J-coupling constants, proton and carbon chemical shifts, NOE distance restraints, dihedral angle restraints starting from a NOE-distance and dihedral-angle refined structure.

The script mini_shift_coup_degen.inp is an example of refinement using J-coupling constants, proton and carbon chemical shifts, NOE distance restraints, dihedral angle restraints, starting from a NOE-distance and dihedral-angle refined structure. Applications with degenerate proton assignments are exemplified by this script.

The script mini_shift_coup_onebond.inp, is an example of refinement using J-coupling constants, proton and carbon chemical shifts, NOE distance restraints, dihedral angle restraints, 1-bond 1JCaHa restraints, starting from a NOE-distance and dihedral-angle refined structure.

An example script including direct secondary carbon chemical shift refinement is test.inp.

Last updated May 05, 1998 at 11:52AM Pacific Daylight Time.