97
6. We found this easiest to do by working out from both domains
and allowing the constructed extended linker model to meet in
the middle. Residues can be added to an existing model in
PyMOL in Editing mode, accessed by clicking the mouse key
shortcuts box in the lower right corner of the viewer window
while in the default Viewing mode. Once in Editing mode,
select the N-terminal nitrogen or C-terminal carbonyl carbon
by clicking on it, then add residues by holding Alt and typing
the single-letter code associated with the desired amino acid.
7. In PyMOL’s Editing mode, select both atoms that should be
bonded in the product, then run PyMOL’s fuse command.
For example if a model with an ECFP on the N terminus of the
protein is desired, start by selecting both the amine of the N
terminus of the protein and the carbonyl carbon of the C terminus of the ECFP. Then, while both atoms remain selected,
enter “fuse” as a command input, which will generate an
approximate model of the fusion protein. The fuse command
may sometimes orient the proteins poorly; make sure to rotate
the proteins as such that they do not overlap in physical space
and the linker is fully extended. This can be done in editing
mode by holding shift and right-clicking on a bond to rotate
the associated torsion angle.
8. For example, you may be working in a subfolder of your home
directory called ~/dyes. You should create new directories for
each conformation, perhaps ~/dyes/open and ~/dyes/closed.
You add the script and starting structure to each directory, and
are left with the following files:
~/dyes/open/open-start.pdb
~/dyes/open/simulations.sh
~/dyes/closed/closed-start.pdb
~/dyes/closed/simulations.sh
9. The script will then generate folders for setup, each run, and
the trimmed, fitted trajectories as pdb files:
~/dyes/open/setup/
~/dyes/open/run1/, ~/dyes/open/run2/,
~/dyes/open/run3/ etc.
~/dyes/open/results/
10. The version numbers given in the materials are known to
work; other versions will probably work as well but have not
been tested. Note that MARTINIZE.py, which is downloaded and run by simulations.sh, is not compatible with
Python 3, and thus Python 2 must be available on your system for the setup steps. The typical name for the DSSP executable varies from system to system; simulations.sh can be
told the correct name for your system either by editing the
dssp_name variable or by passing the correct name as an argument
Synthetic-Dye Fluorescent Protein FRET Sensors
6. We found this easiest to do by working out from both domains
and allowing the constructed extended linker model to meet in
the middle. Residues can be added to an existing model in
PyMOL in Editing mode, accessed by clicking the mouse key
shortcuts box in the lower right corner of the viewer window
while in the default Viewing mode. Once in Editing mode,
select the N-terminal nitrogen or C-terminal carbonyl carbon
by clicking on it, then add residues by holding Alt and typing
the single-letter code associated with the desired amino acid.
7. In PyMOL’s Editing mode, select both atoms that should be
bonded in the product, then run PyMOL’s fuse command.
For example if a model with an ECFP on the N terminus of the
protein is desired, start by selecting both the amine of the N
terminus of the protein and the carbonyl carbon of the C terminus of the ECFP. Then, while both atoms remain selected,
enter “fuse” as a command input, which will generate an
approximate model of the fusion protein. The fuse command
may sometimes orient the proteins poorly; make sure to rotate
the proteins as such that they do not overlap in physical space
and the linker is fully extended. This can be done in editing
mode by holding shift and right-clicking on a bond to rotate
the associated torsion angle.
8. For example, you may be working in a subfolder of your home
directory called ~/dyes. You should create new directories for
each conformation, perhaps ~/dyes/open and ~/dyes/closed.
You add the script and starting structure to each directory, and
are left with the following files:
~/dyes/open/open-start.pdb
~/dyes/open/simulations.sh
~/dyes/closed/closed-start.pdb
~/dyes/closed/simulations.sh
9. The script will then generate folders for setup, each run, and
the trimmed, fitted trajectories as pdb files:
~/dyes/open/setup/
~/dyes/open/run1/, ~/dyes/open/run2/,
~/dyes/open/run3/ etc.
~/dyes/open/results/
10. The version numbers given in the materials are known to
work; other versions will probably work as well but have not
been tested. Note that MARTINIZE.py, which is downloaded and run by simulations.sh, is not compatible with
Python 3, and thus Python 2 must be available on your system for the setup steps. The typical name for the DSSP executable varies from system to system; simulations.sh can be
told the correct name for your system either by editing the
dssp_name variable or by passing the correct name as an argument
Synthetic-Dye Fluorescent Protein FRET Sensors