#Xwalk (version 0.6)

##INTRODUCTION Chemical cross-linking of proteins or protein complexes and the mass spectrometry based localization of the cross-linked amino acids is a powerful method for generating distance information on the substrate topology. Here we introduce the algorithm Xwalk for predicting and validating these cross-links on existing protein structures. Xwalk calculates and displays non-linear distances between chemically cross-linked amino acids on protein surfaces, while mimicking the flexibility and non-linearity of cross-linker molecules. It returns a Solvent Accessible Surface (SAS) distance, which corresponds to the length of the shortest path between two amino acids, where the path leads through solvent occupied space without penetrating the protein surface.

#TEST Test examples to execute Xwalk can be found in the test subdirectory.

#CLASSPATH You might want to consider adding the bin directory into the CLASSPATH environment of your SHELL, which avoids the usage of the -cp flag when executing Xwalk.

#COMMANDLINE PARAMETERS A list of all commandline parameters can be retrieved by typing -help before a Xwalk execution.

#OUTPUT Distance information will be printed out to the STDOUT channel or via -out to a local file in the following tab delimeted format:

1	1brs.pdb	LYS-1-D-CB	LYS-2-D-CB	1	5.9	6.6	0.0449	0.0685	KKAVINGEQIR-KAVINGEQIR

Column	Information
1	Index
2	Filename
3	PDB information of the 1st amino acid in the format: PDBThreeLetterCode-ResidueId-ChainId-AtomName
4	PDB information of the 2nd amino acid in the format: PDBThreeLetterCode-ResidueId-ChainId-AtomName
5	Sequence distance within the PDB file as calculated from the residue identifiers of the 1st and 2nd amino acid.
6	Euclidean distance between 1st and 2nd amino acid.
7	SAS distance between 1st and 2nd amino acid.
8	Probability for observing the Euclidean distance in a XL experiment using DSS or BS3.
9	Probability for observing the SAS distance in a XL experiment using DSS or BS3.
10	Shortest tryptic peptide sequence for both amino acids.

Setting -pymol -out xxx.pml on the commandline will write a PyMOL script into the file xxx.pml, which can subsequently be loaded into the molecular viewer PyMOL to visualise SAS distance paths (see NOTES).

#ALGORITHM The SAS distance is calculated using a grid and the breadth-first search algorithm to search within the grid for the shortest path between two points on the protein surface using following algorithm:

1. Read in input data
   1. xyz.pdb spatial coordinates of a protein or protein complex in PDB format.
   2. maxdist maximum distance of the path (i.e. the length of the cross-linker + AA side chain length)
   3. listXL a list of experimentally determined cross-linked lysine residues.
2. Remove all non-protein atoms in xyz.pdb and assign protein atoms a van der Waals radius sum of SURFNET atom radii + solvent radius
3. Select a random lysine pair (K_i,K_j) from listXL,
4. Check Euclidean distance (Euc) of (K_i,K_j). Continue, if Euc > maxdist, disregard otherwise and go back to 3.
5. Generate a grid of size maxdist and grid spacing 1 Angstroem centered at AAa
6. Set Integer.MAX_VALUE as distance for all grid cells and label grid cells as residing in the
7. protein
8. solvent
9. boundary between protein and solvent.
10. Label grid cells residing in (K_i,K_j) as solvent
11. Set distance dist = 0.0 for central grid cell of K_i and store grid cell in the active list listactive
12. Start breadth-first search. Iterate through listactive
13. Check that grid cell i is labeled as solvent
14. Find all immediate neighbors listneighbour
15. Iterate through listneighbour 1. Check that grid cell j is labeled as solvent 2. Compute new distance for grid cell j as the sum of the distance in grid cell i and the Euclidean distance between grid cell i and j 3. If distance sum in 9.3.2 is smaller than the current distance in grid cell j, store the distance sum as new distance for grid cell j and add grid cell j to the new active list listnew_active,
16. Go back to step 9. with listactive = listnew_active.

#NOTES

• As the SAS distance is based on a grid calculation, the default heap size of the Java VM with 64MB is likely to be too small. You can increase the heap size with java -Xmx512m
• You can obtain PyMOL here for free.
• Beware that in order for PyMOL to recognize the script file, the file must have the name ending .pml.
• You can load the script directly at the startup of PyMOL, i.e. with the command

  pymol 1brs.pml.
  

#CONTACT [email protected]

#LICENCE Xwalk executable and libraries are available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License via the Xwalk website.

• Anyone is free:

  • to copy, modify, distribute the software;

• Under the following conditions:

  • the original authors must be given credit by citing the original Xwalk paper:
    • Kahraman A., Malmström L., Aebersold R. (2011). Xwalk: Computing and Visualizing Distances in Cross-linking Experiments. Bioinformatics, doi:10.1093/bioinformatics/btr348.
  • the software or derivate works must not be used for commercial purposes
  • derivate works must be licenced under the same or similar licence

• Any of the above conditions can be waived if the authors give permission.

VERSION HISTORY

0.6

• Added parameter -bfactor, which adds the uncertainty of the atom coordinates as expressed by their B-factor/temperature factor to the maximum distance threshold.
• Distance probabilities were updated based on the latest update of XLdb with 506 intra-protein and 62 inter-protein cross-links.
• Added Electro.java to the utils package for calculating electrostatic potentials on molecular surfaces. Requires APBS, PDB2PQR and SIMS.
• Added Compactness.java to the utils package for calculating the number of atoms within a certain radius of any atom in a protein.
• Bug fixes.

###0.5

• Added parameter -xSC, which is similar to -bb except that it leaves all side chain atoms of non-modified amino acid untouched, while removing only side chain atoms of cross-linked amino acids (except their CB atoms). In addition the solvent probe sphere is kept at the conventional radius of 1.4 Angstroem prior to SAS distance calculations. This parameter might be of value when side chain conformations of cross-linked residues are unknown.
• The premature halt of SAS distance calculation has been removed, which in the previous versions ensured a faster execution time, however, at the cost of the distance calculation accuracy.
• Distance probabilities have been recalculated using a larger data set of published cross-links.
• Bug fixes.

###0.4

• Maximum distance threshold for SAS distance calculation has been increased to 100 Angstroem.
• Added option to read in .gzip files
• Warning messages are output for redundant XL entries in -dist files.
• Added parameter -keepName to keep protein names from -dist files in the output.
• white background colour command in PyMOL scripts has been removed.

###0.3

• Improved performance and memory usage. Xwalk runs now 3-5 times faster, while using a third of the previous memory.

0.2.1

• Fixed a bug that caused Xwalk to crash on cases where cross-linked atoms were located outside the grid.
• Added a further number code to the SAS distance column for atom pairs that are located in closed cavities prohibiting a shortest path calculation.
• Bug fixes

###0.2

• Probability calculation with option -prob
• Number code in SAS distance column (see -help)
• Euclidean distance for all residue pairs in a distance file (-dist)
• PyMol option changed from -p to -pymol
• Removed -grid, -global and -all commandline options
• Bug fixes

###0.1

• First version of Xwalk