4. Since no density maps are available for the tryptophan synthase
complex, a simulated map at 14 A ˚ “TS_1WBJ_14A.mrc” has
been generated and included in the protocol files.
5. All subunits will need to be fitted into the map of the density
for the map to restrain the subunit positions during sampling.
For this protocol, the structure has been pre-fitted into the mrc
map (see Notes 9 and 10).
6. The mrc density map will need to be converted into a
modeling-compatible Gaussian Mixture Model (GMM)
format.
7. To do this, have the “initial_map.mrc,” “calculate_density.sh”
and “create_gmm.py” files in the same directory, and open a
terminal window, e.g., Terminal in MacOS/Linux or Bash for
Windows.
8. Enter “bash create_gmm.py initial_map.mrc 6” to automatically begin converting the simulated mrc map into a GMM map
(see Notes 11 and 12). The number at the end refers to the
number of components that the map will possess—the higher
the value, the finer the resolution of the generated map model.
The files “initial_map.gmm.n6.mrc” and “initial_map.gmm.
n6.txt” will be generated. Optionally open the new and original maps to visualize the conversion.
3.6 Preparing
Modeling Script
for IMP
1. Open the topology.txt file which has already been filled
(Fig. 7a).
2. The topology file contains a “topology_dictionary” for each
subunit. Table 1 explains the function of each heading.
3. In the previous directory, there will be a script called “modelling.py” which will call IMP and perform the modeling.
4. “modelling.py” is a python script that will instruct IMP on how
to initialize each subunit and perform random sampling while
restraining their positions and orientations using the stoichiometry, cross-link, and EM restraints.
5. Within “modelling.py,” lines 25–27 under the “Define Input
Files” section will link IMP to the necessary “topology.txt” and
“initial_map.gmm.n6.txt” files which are located in the “./
inputs/” directory (Fig. 7b).
6. The next lines “num_frames” and “num_mc_steps” will set the
number of iterations that the sampling will be performed for
and the number of Monte Carlo steps that are included in one
iteration.
7. “rb_max_trans” and “rb_max_rot” are variables for controlling
the step size of each translation and rotation of each rigid body,
i.e., each subunit.
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complex, a simulated map at 14 A ˚ “TS_1WBJ_14A.mrc” has
been generated and included in the protocol files.
5. All subunits will need to be fitted into the map of the density
for the map to restrain the subunit positions during sampling.
For this protocol, the structure has been pre-fitted into the mrc
map (see Notes 9 and 10).
6. The mrc density map will need to be converted into a
modeling-compatible Gaussian Mixture Model (GMM)
format.
7. To do this, have the “initial_map.mrc,” “calculate_density.sh”
and “create_gmm.py” files in the same directory, and open a
terminal window, e.g., Terminal in MacOS/Linux or Bash for
Windows.
8. Enter “bash create_gmm.py initial_map.mrc 6” to automatically begin converting the simulated mrc map into a GMM map
(see Notes 11 and 12). The number at the end refers to the
number of components that the map will possess—the higher
the value, the finer the resolution of the generated map model.
The files “initial_map.gmm.n6.mrc” and “initial_map.gmm.
n6.txt” will be generated. Optionally open the new and original maps to visualize the conversion.
3.6 Preparing
Modeling Script
for IMP
1. Open the topology.txt file which has already been filled
(Fig. 7a).
2. The topology file contains a “topology_dictionary” for each
subunit. Table 1 explains the function of each heading.
3. In the previous directory, there will be a script called “modelling.py” which will call IMP and perform the modeling.
4. “modelling.py” is a python script that will instruct IMP on how
to initialize each subunit and perform random sampling while
restraining their positions and orientations using the stoichiometry, cross-link, and EM restraints.
5. Within “modelling.py,” lines 25–27 under the “Define Input
Files” section will link IMP to the necessary “topology.txt” and
“initial_map.gmm.n6.txt” files which are located in the “./
inputs/” directory (Fig. 7b).
6. The next lines “num_frames” and “num_mc_steps” will set the
number of iterations that the sampling will be performed for
and the number of Monte Carlo steps that are included in one
iteration.
7. “rb_max_trans” and “rb_max_rot” are variables for controlling
the step size of each translation and rotation of each rigid body,
i.e., each subunit.
Mass Spectrometry-Based Protein Modelling
231