initial_map.gmm.nX.txt—Gaussian mixture model (GMM)
representation of the electron density map.
topology.txt—contains topological parameters for initializing
each subunit for modeling.
Scripts
modelling.py—script for calling IMP.
calculate_density.sh—bash script for converting density maps
to modeling-compatible maps.
create_gmm.py—IMP script for generating the above maps.
2. The following steps will walk through how to prepare these
files.
3.2 Sourcing
Structures
of Constituent
Subunits
1. Subunits of the complex may be available as crystal or NMR
structures; these may be found at repositories such as RCSB
and PDBe. A crystal structure of the tryptophan synthase
complex has been deposited under PDB 1WBJ (https://www.
rcsb.org/structure/1WBJ). Using PyMOL, enter the “fetch
1WBJ” command into the PyMOL command line to automatically download the file from the RCSB database (see Note 1).
2. PyMOL will display two subunits: alpha (chain A) and beta
(chain B). Display the sequence information by pressing the
“S” button displayed on the bottom right hand side of the
Movies Controls or by entering “set seq_view, on” into the
PyMOL command line.
3. The tryptophan synthase dimer will be coated with ligands and
water molecules which will need to be removed prior to modeling. Type “remove hetatm” into the PyMOL command line to
remove all non-protein atoms.
4. Optionally, type “util.cbc” to enable per-chain coloration to aid
visualization of the subunits. The alpha subunit (chain A) will
be colored green, and beta subunit (chain B) in blue by default.
A screenshot of your PyMOL GUI at this point is shown in
Fig. 2a.
5. With the sequence view on, check the following:
Sequence numbering—check the sequence numbering to
verify that the numbers are correct. Additionally, locate on the
structure the residues involved in cross-linking and verify that
their residue numbers are correct, e.g., check that residue
LYS46 on the cross-link list correctly points to LYS46 on the
structure (see Note 2).
Residue type—only certain residue types will be “visible”
in the experiment depending on the type of cross-linker used,
e.g., BS3 (bis(sulfosuccinimidyl)suberate) is an amine-toamine cross-linker only targets lysine and N-terminal amine
Mass Spectrometry-Based Protein Modelling
225
representation of the electron density map.
topology.txt—contains topological parameters for initializing
each subunit for modeling.
Scripts
modelling.py—script for calling IMP.
calculate_density.sh—bash script for converting density maps
to modeling-compatible maps.
create_gmm.py—IMP script for generating the above maps.
2. The following steps will walk through how to prepare these
files.
3.2 Sourcing
Structures
of Constituent
Subunits
1. Subunits of the complex may be available as crystal or NMR
structures; these may be found at repositories such as RCSB
and PDBe. A crystal structure of the tryptophan synthase
complex has been deposited under PDB 1WBJ (https://www.
rcsb.org/structure/1WBJ). Using PyMOL, enter the “fetch
1WBJ” command into the PyMOL command line to automatically download the file from the RCSB database (see Note 1).
2. PyMOL will display two subunits: alpha (chain A) and beta
(chain B). Display the sequence information by pressing the
“S” button displayed on the bottom right hand side of the
Movies Controls or by entering “set seq_view, on” into the
PyMOL command line.
3. The tryptophan synthase dimer will be coated with ligands and
water molecules which will need to be removed prior to modeling. Type “remove hetatm” into the PyMOL command line to
remove all non-protein atoms.
4. Optionally, type “util.cbc” to enable per-chain coloration to aid
visualization of the subunits. The alpha subunit (chain A) will
be colored green, and beta subunit (chain B) in blue by default.
A screenshot of your PyMOL GUI at this point is shown in
Fig. 2a.
5. With the sequence view on, check the following:
Sequence numbering—check the sequence numbering to
verify that the numbers are correct. Additionally, locate on the
structure the residues involved in cross-linking and verify that
their residue numbers are correct, e.g., check that residue
LYS46 on the cross-link list correctly points to LYS46 on the
structure (see Note 2).
Residue type—only certain residue types will be “visible”
in the experiment depending on the type of cross-linker used,
e.g., BS3 (bis(sulfosuccinimidyl)suberate) is an amine-toamine cross-linker only targets lysine and N-terminal amine
Mass Spectrometry-Based Protein Modelling
225