15. A PyMOL script has been provided to streamline the conversion between coarse-grained and atomistic models, called
“CG2pdb.py” in the same directory as “modelling.py.”
16. Open a terminal window and direct to the directory of
“CG2pdb.py.” Run the script using “-c CG2pdb.py.” The will need to be
changed to the path of PyMOL on your system. The “-c” flag
will suppress the GUI of PyMOL.
17. The directory “output_atomistic” will be made and will contain all 100 best scoring IMP models in atomistic representation. By default, the CG2pdb.py script will convert from
model.0.pdb to model.99.pdb (100 models). You may alter
the model range within the script by changing “n_first” and
“n_last” variables.
18. At this point, you will have a folder called “output_atomistic”
containing 100 atomistic models of tryptophan synthase that
have been generated using modeling restraints from MS
and EM.
3.8 Calculating
the CCS of Generated
Models
1. The theoretical collision cross section (CCS) of atomic models
can be calculated using software such as IMPACT [43] (see
Notes 17–19).
2. This value can be compared to experimentally determined CCS
via IM-MS [44].
3. Alternatively, CCS values for complexes may be found in published literature [7].
4. The CCS of the tryptophan synthase complex is approximately
7200 A ˚ 2 .
5. We will use IMPACT to calculate the projection approximation
(PA) of each model.
6. Open a terminal window and direct to the “output_atomistic”
folder.
7. With IMPACT callable from your PATH variable, run “impact
*.pdb -o output_file_CCS.txt.” *.pdb will tell IMPACT to run
through file with the .pdb file extension.
8. When IMPACT has finished, output_file_CCS.txt will display a
list of each file, with the calculated PA and TJM values (see
Note 20).
9. Linear scaling of the calculated PA by an empirical factor of
1.14 has been shown to provide a good approximation of the
true model CCS [45].
10. “output_file_CCS.txt” can be opened using spreadsheet software, and a scaling factor of 1.14 can be applied to each value
under “CCS_PA.”
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235
“CG2pdb.py” in the same directory as “modelling.py.”
16. Open a terminal window and direct to the directory of
“CG2pdb.py.” Run the script using “
changed to the path of PyMOL on your system. The “-c” flag
will suppress the GUI of PyMOL.
17. The directory “output_atomistic” will be made and will contain all 100 best scoring IMP models in atomistic representation. By default, the CG2pdb.py script will convert from
model.0.pdb to model.99.pdb (100 models). You may alter
the model range within the script by changing “n_first” and
“n_last” variables.
18. At this point, you will have a folder called “output_atomistic”
containing 100 atomistic models of tryptophan synthase that
have been generated using modeling restraints from MS
and EM.
3.8 Calculating
the CCS of Generated
Models
1. The theoretical collision cross section (CCS) of atomic models
can be calculated using software such as IMPACT [43] (see
Notes 17–19).
2. This value can be compared to experimentally determined CCS
via IM-MS [44].
3. Alternatively, CCS values for complexes may be found in published literature [7].
4. The CCS of the tryptophan synthase complex is approximately
7200 A ˚ 2 .
5. We will use IMPACT to calculate the projection approximation
(PA) of each model.
6. Open a terminal window and direct to the “output_atomistic”
folder.
7. With IMPACT callable from your PATH variable, run “impact
*.pdb -o output_file_CCS.txt.” *.pdb will tell IMPACT to run
through file with the .pdb file extension.
8. When IMPACT has finished, output_file_CCS.txt will display a
list of each file, with the calculated PA and TJM values (see
Note 20).
9. Linear scaling of the calculated PA by an empirical factor of
1.14 has been shown to provide a good approximation of the
true model CCS [45].
10. “output_file_CCS.txt” can be opened using spreadsheet software, and a scaling factor of 1.14 can be applied to each value
under “CCS_PA.”
Mass Spectrometry-Based Protein Modelling
235