AutoRTM

AutoRTM is a software package to determine a 3D reconstruction from a single migrograph of icosahedral particles. The approach is fully automated and it is intended to be used during a microscopy session, to gain structural information from a sample while being initially evaluated.

The program is provided with a graphical interface for easy configuration and data inspection, and builds upon the capabilities of Auto3DEM to perform the computations. Once started and configured, the program monitors a target directory, and process each micrograph found as soon as available. Steps performed are CTF determination, particle localization and 3D reconstruction (using the Random Model Computation method). All this information is displayed in the graphical interface. For further information please refer to the article cited below.

• The installation procedure is valid both for Linux and Mac OSX systems.
• Download the file autortm_151124.tar.gz and uncompress it

tar xvfz autortm_151124.tar.gz

• Go inside the directory created and launch the installation, specifying the final directory where to install the program. The directory can not exist at the launch of the procedure

cd autortm_build
./autortm_install <install dir>

• At the end of the procedure, follow the instructions on terminal on how to set the shell environment
• Delete the build directory

cd ../
rm -rf autortm_build

The procedure will install a complete python environment, with the all the modules and libraries required by the program.

The procedure will also install two external software packages, Bsoft (ver. 1.9.0) and Auto3DEM (ver. 4.5.2). If, for some reason, the installation procedure is not able to install these packages or to configure the environment to access them from the program, as an alternative you can install the two software manually (they are available in the a3dm and bsoft subdirectories), and then launch the installation with the options –skip-bsoft and –skip-auto3dem.

Finally the program relies on Chimera to visualize 3D models. Therefore the installation procedure will try to determine the path to the Chimera executalbe. In case it is not able to find it but you have it, you have two options

1. set the path to the chimera binary in the cpath variable inside the file src/session.py in your installation directory
2. save a default session configuration file and modify its parameter chimera_path

The program is launched from a terminal using the command autortm. It displays a graphical interface from where to launch a processing session and monitor the progress of the processing. From the interface you can access to a session menu where you can set up an acquisition before starting the processing.

A session contains all the information to process a specific sample. The configuration can be saved in a session file (.ars extension) for future use. Critical parameters are the microscope settings (voltage, spherical aberration and pixel size) and the information on the sample (amplitude contrast and approximate diameter of the particle). Depending on the computing system you are using, you can also modify the number of processors used for different tasks during the analysis. A special option, Run multiple models in parallel, allows to run multiple model computations in parallel, but it only works on systems that use the Torque/PBS queue system to launch multiple jobs. Additional parameters determining a session, such as the version of CTFFIND to use (default: 3, version 4 currently available only on Linux systems) or the path to the Chimera executable, can only be modified in the .ars session file, using a text editor.

Once a session is properly set up, from the main interface it is possible to specify the Directory to monitor, that is the directory where the acquisition system controlling the microscope will store the images acquired, and the Directory for processing, where all the results from the analysis will be stored. At this point you can press the button Start monitoring, and the program will start to process each micrograph acquired as soon as it is stored in the given input directory. At any point you can stop the processing of further images by pressing the same button (now Stop monitoring): if at that moment a micrograph is being processed, the program will still complete its analysis. To remark that the program only processes micrographs that have not been previously analyzed: in other words, if the directory for processing already contains the results of a previous analysis from one micrograph, this micrograph will not be analyzed again, instead the results already available will be displayed in the GUI.

In order to get familiar with the program, here is provided a Test Dataset to download. The compressed archive contains few micrographs of Giardia lamblia virus (GLV) (see Janssen M.E. et al (2015) J. Virol. 89:1182-94 http://dx.doi.org/10.1128/jvi.02745-14 for details on the virus and the microscopy) and two session files, GLVdataset.ars and GLVdatasetctf4.ars (for using CTFFIND4, only valid on Linux), with all the required settings. To test the program:

• uncompress the archive and go into the directory generated

tar xvfz TestCaseAutoRTM_GLV.tar.gz

• go into the directory generated and launch the program

cd GLV
autortm

• from the Session menu, load one of the session file and then press the button Start monitoring.
• inspect the results as displayed after the processing of each micrograph.

The program was written mostly as a proof-of-concept for real-time, high-throughput single particle microscopy. Currently it is not actively developed, and only minimal support is provided. If you have questions, please contact the authors.

AutoRTM is free to use for educational, research and non-profit purposes (see LICENSE).

Development of AutoRTM has been funded by NIH Grant R01-GM087708 (2010-2014).

The software was developed by Giovanni Cardone and Xiaodong Yan.

If you find this software useful for your research project, please cite:

Cardone G., X. Yan, R. S. Sinkovits, J. Tang, and T. S. Baker (2013) Three-dimensional reconstruction of icosahedral particles from single micrographs in real time at the microscope. J. Struct. Biol. 183:329-341. (http://dx.doi.org/10.1016/j.jsb.2013.07.007)