Crosslinking the E.coli replisome

Figure 1 The E. coli replisome mapped with data analysed by Hekate. The elucidation of the exonuclease's tail (yellow) wrapping round the polymerase (orange) was a key result of this study.3

Hekate

Hekate is a suite of tools that address the challenges involved in analysing protein cross-linking experiments when combined with mass spectrometry. The software is an integrated pipeline for the automation of the data analysis workflow and provides a novel scoring system based on principles of linear peptide analysis. In addition, it provides a tool for the visualization of identified cross-links using three-dimensional models, which is particularly useful when combining chemical cross-linking with other structural techniques.

The results of this work were published as:

  1. Holding et. al (2013) J. Proeome Res. 2013. [PubMed]
  2. Liu et. al (2013) Genes Dev. 2013. [PubMed]
  3. Rego et. al (2013) EMBO J. 32. 1334-1343. [Pubmed] [F1000Prime]

How it works

Hekate works with and without the use of isotopic labels to identify possible cross-linked peptides. The peptides are assigned a sequence using an algorithm based on that previously described by Cox et al. for the scoring of linear peptides. Data is submitted to the software in Mascot Generic Format (mgf) and it should be able to analyse the output of most types of mass spectrometer used for proteomic work.


Comparison of Hekate and Mascot Scoring

Figure 2 Comparision of Hekate and Mascot scores on linear peptides. As with previous studies, the stastical method used is found to correlate well with Mascot on the assignment of linear peptides, allowing for confidence in the results.

Result confidence can futher be monitored by the use of a False Discovery Rate (FDR) which can optionally be generated by use of a decoy search.

Obtaining

Hekate can be obtained in two ways. As the source code (which is a set of perl scripts) from the Hekate repository on GitHub (locally mirrored) which is more suitible for those who have experience of installing software on a webserver. The second option is to download it as a Virtualbox compatible machine image containing Hekate preinstalled on Ubuntu. The later will allow you to run Hekate on you desktop as a virtual server and is probably the most simple way of testing the software. The username and password for the default account on this virtual machine are both 'hekate'.


Installing

Hekate as a Virtual Machine

A virtual machine is like having a computer running in a window on your desktop. This can be helpful when you don't have a computer spare to run as a server, or you have little experience of Linux and installing webservers as it gives you a safe environment to try things out. Hekate is provided as a VirtualBox image for this purpopse. This allows you to download a pre-installed copy of the software that you can run on your computer locally, quickly and without too much difficulty.

  1. Download and install Oracle VM Virtual Box, which is availiable for free from their website.

  2. Download a copy of the Hekate Virtual Machine to your computer.

  3. Open the downloaded file 'hekate.ova' in VirtualBox (the easiest way to do this is usually to double click the file) and import it into VirtualBox. The default settings should be fine.

  4. You now have an installed copy of Hekate on a Virtual Machine. Tell VirtualBox to boot the virtual machine by clicking 'start'.

  5. Once the virtual machine has booted you should be presented with the Ubuntu desktop. Firefox is already installed and Hekate is set as the homepage, making it easy to access from the virtual machine's desktop.

  6. The default settings will configure the webserver to be accessible from the your computer. The address http://localhost:8080 should allow you to access it from outside the virtual machine on the host computer.


Hekate running on a virtual machine.


Figure 3 Hekate running on a virtual machine and being accessed externally by the host machine's web browser.


Hekate on a Server

This is a brief guide to how to install Hekate and assumes a working knowledge of Linux. It is recommended that Hekate is run on a fresh Debian Linux installation and the the server is not accessible via the internet.

These instructions are currently out of date. For now the recommended route is the virtual machine provided above, however experianced Linux users should have no problem installing by adapting these steps.

  1. Install the required packages that are not included in the standard installation of your system. For Debian these are:

    • apache2
    • git
    • libparallel-forkmanager-perl
    • libdbd-sqlite3-perl
    • libchart-gnuplot-perl

    These can be installed with 'apt-get' or 'aptitude'.

    apt-get install apache2 
    
  2. Make and change to directory '/srv/www'.

    mkdir /srv/www
    cd /srv/www
    
  3. Use Git to obtain the latest version of Hekate and download it into the current directory.

    git clone git://github.com/AndrewHolding/Crosslinker
    
  4. Change ownership of the folder to www-data with chown.

    chown www-data:www-data /srv/www -R
    
  5. Update Apache's default site file or create a new site definition to point to the Hekate install.

    nano /etc/apache2/sites-available/default
    
    Change following lines:
    
    DocumentRoot /var/www to DocumentRoot /srv/www/Hekate/html
    
    <Directory /var/www/> to <Directory /srv/www/Hekate/html>
    
    ScriptAlias /cgi-bin/ /usr/lib/cgi-bin/
    <Directory "/usr/lib/cgi-bin">
    
        to      
    
    ScriptAlias /cgi-bin/ /srv/www/Hekate/cgi-bin/
    <Directory "/srv/www/Hekate/cgi-bin">
    
  6. Restart the web server.

    /etc/init.d/apache2 restart
    
  7. You should be now able to access Hekate by connecting to the server with a web browser.

    http://localhost/