tem-simulator-scripts

This repository contains convenient scripts for the TEM-Simulator V1.3 (http://tem-simulator.sourceforge.net/)

Installation

conda create -n temscripts -c conda-forge python=3.9 biotite
conda activate temscripts
pip install tem-simulator-scripts

Make also sure that IMOD is installed, as the tsimscripts_pipe.sh needs the commands submfg and trimvol from IMOD.

Preparation

Download all PDBs you want to simulate. In case PDB is not available (only PDBx or mmcif), download PDBX or mmcif and convert it to PDB with https://mmcif.pdbj.org/converter/index.php?l=en . Its recommended to download directely the biological assemblies.

Overview

Here we give a short overview of the scripts contained in this package:

• tsimscripts_pipe.sh: This is probably the only script a user needs from this collection.
• tsimscripts_gen_coords.py: Generates coordinates for particles that are not overlapping.
• tsimscripts_gen_input.py: This script generates the input file for the TEM-Simulator.
• tsimscripts_gen_filaments.py: Generate simulated filaments.
• tsimscripts_gen_raw_tilt.py: Generates the raw tilt file for IMOD.
• tsimscripts_gen_trans_file.py: Some PDBs needs transformation files. Simply use assemblies to avoid this script.
• tsimscripts_gen_map.py: Script to generates vesicle or fiducial maps.
• tsimscripts_extract.py: Extracts subvolumes of the simulated tomogram.

Pipeline all commands

A convenient script is fully automating the whole process incl. reconstruction.

It requires that imod is installed. The require files for simulating filaments can be found in resources/filament_files

A sample command looks like this:

tsimscripts_pipe.sh --pdbs pdbs/*.pdb --npdbs 150 --output out_sim_tomo_1 --random_seed 10 --pdbs_fil filament_files/*.pdb --settings_fil filament_files/*.json --nsubs 100 --dose 15000 --random_seed 10

It will generate a tomogram with all pdbs included in the folder pdbs/ and will add 4 vesicles and 10 fiducials.

How to simulate a tomogram with particles from a single PDB

You can also run each step individually in case you want to have more control:

0. Install the package with python setup.py install
1. Optional: Generate trans files using tsimscripts_gen_trans_file.py. Not necessary when using biological assemblies directly.
2. Optional: Generate fiducial using tsimscripts_gen_map.py: I would add 8-10 fiduicals. Patch tracking didnt work well.
3. Generate coordinate tsimscripts_gen_coords.py
4. Generate input file using tsimscripts_gen_input_file.py
5. Generate raw tilt file with tsimscripts_gen_raw_tilt.py
6. Run TEM-simulator input.txt
7. Reconstruction with etomo
   • During Coarse Alignement I needed to use a low freq. rolloff sigma of 0.01, a high freq. cutoff sigma of 0.25 and a high freq. rolloff sigma of 0.05. Moreover I activated the option No Cosine Stretch.
   • In Final Aligned Stack Complete I set the binning to 2
   • In Tomogram Generation In Progress I set the the thickness in Z to 300
8. Extract the subvolumes with tsimscripts_extract.py: If you have used fiducials, check if they are centered in the extracted tomograms. If they offcentered, repeat the alignement in step 7.

How to simulate a tomogram with particles from multiple PDBs

Here is a complete example how to simulate a tomogram. It assumes that there are several pdbs in the folder pdbs/. Only the PDB 6x9q needed a trans_file in this example.

tsimscripts_gen_map.py fiducial -d 50 --apix 1 -v 10000
mkdir trans_files
tsimscripts_gen_trans_file.py pdbs/6x9q.pdb
mv 6x9q_trans.txt trans_files/
tsimscripts_gen_coords.py --pdbs pdbs/*.pdb --npdbs 20 -o coords/
tsimscripts_gen_input.py --pdbs pdbs/*.pdb fiducial.mrc --trans trans_files/6x9q_trans.txt --coords coords/*.txt --defocus_upper 6 --defocus_lower 7
TEM-simulator input.txt