interest, and display it in line or stick format. To do this, select
the residue by clicking on it in the sequence viewer, click on “S”
in the Object Control Panel for “(sele)” and press either “lines”
or “sticks” (Fig. 2b).
6. Next, you will need to identify any missing residues in each of
the subunits. This can be done by aligning the sequence of each
subunit to their canonical sequences (from UniProt) using a
multiple sequence alignment program such as T-Coffee (see
Notes 4 and 5).
7. First, save the 1WBJ PDB file in the FASTA format in PyMOL
by entering both “save initial_model_A.fasta, chain A” and
“save initial_model_B.fasta, chain B” into the PyMOL command line. This will save the sequences of both chain A (alpha
subunit) and chain B (beta subunit) separately as FASTA files.
8. Access the UniProt entry of the tryptophan synthase alpha
subunit (Accession Number (AN): P00929) and beta subunit
(AN: P0A2K1) at: https://www.uniprot.org/uniprot/
P00929 and https://www.uniprot.org/uniprot/P0A2K1,
respectively.
9. The sequence of each subunit is listed under the “Sequence”
tab of the side bar. Click on the “FASTA” download button to
open the sequence in the FASTA format.
10. Next, we will align the model and canonical sequences using
the T-Coffee webserver. Copy and paste the model and canonical sequences of the alpha chain into T-Coffee and submit.
Repeat for the beta chain. Sample results are shown in Fig. 3a.
11. Regions with differences between theoretical and modeled
sequences will be identified by sequence alignment, including
missing residues (Fig. 3a).
12. If there are many regions of the model with missing coordinates, homology modeling may need to be employed to regenerate these missing residues (see Note 6).
13. Finally, combine the “initial_model_A.fasta” and “initial_model_B.fasta” sequences into a single “initial_model.fasta.”
Since there are two copies of alpha and beta subunits in the
complex, duplicate both the alpha and beta subunit sequences,
and title each subunit as “>TS_alpha_A,” “>TS_beta_B,”
“>TS_alpha_C,” and “>TS_beta_D” (Fig. 3b).
3.3 Determining
Complex
Stoichiometry Using
Native MS
1. The native MS spectra of the tryptophan synthase complex can
be used to determine any subcomplexes that build up the full
complex, and the mass of these subcomplexes (Fig. 4).
2. A native MS spectra of the complex can be found at [33].
3. Use the subcomplexes to determine the known connections
between the constituent subunits (see Notes 7 and 8). The
Mass Spectrometry-Based Protein Modelling
227
the residue by clicking on it in the sequence viewer, click on “S”
in the Object Control Panel for “(sele)” and press either “lines”
or “sticks” (Fig. 2b).
6. Next, you will need to identify any missing residues in each of
the subunits. This can be done by aligning the sequence of each
subunit to their canonical sequences (from UniProt) using a
multiple sequence alignment program such as T-Coffee (see
Notes 4 and 5).
7. First, save the 1WBJ PDB file in the FASTA format in PyMOL
by entering both “save initial_model_A.fasta, chain A” and
“save initial_model_B.fasta, chain B” into the PyMOL command line. This will save the sequences of both chain A (alpha
subunit) and chain B (beta subunit) separately as FASTA files.
8. Access the UniProt entry of the tryptophan synthase alpha
subunit (Accession Number (AN): P00929) and beta subunit
(AN: P0A2K1) at: https://www.uniprot.org/uniprot/
P00929 and https://www.uniprot.org/uniprot/P0A2K1,
respectively.
9. The sequence of each subunit is listed under the “Sequence”
tab of the side bar. Click on the “FASTA” download button to
open the sequence in the FASTA format.
10. Next, we will align the model and canonical sequences using
the T-Coffee webserver. Copy and paste the model and canonical sequences of the alpha chain into T-Coffee and submit.
Repeat for the beta chain. Sample results are shown in Fig. 3a.
11. Regions with differences between theoretical and modeled
sequences will be identified by sequence alignment, including
missing residues (Fig. 3a).
12. If there are many regions of the model with missing coordinates, homology modeling may need to be employed to regenerate these missing residues (see Note 6).
13. Finally, combine the “initial_model_A.fasta” and “initial_model_B.fasta” sequences into a single “initial_model.fasta.”
Since there are two copies of alpha and beta subunits in the
complex, duplicate both the alpha and beta subunit sequences,
and title each subunit as “>TS_alpha_A,” “>TS_beta_B,”
“>TS_alpha_C,” and “>TS_beta_D” (Fig. 3b).
3.3 Determining
Complex
Stoichiometry Using
Native MS
1. The native MS spectra of the tryptophan synthase complex can
be used to determine any subcomplexes that build up the full
complex, and the mass of these subcomplexes (Fig. 4).
2. A native MS spectra of the complex can be found at [33].
3. Use the subcomplexes to determine the known connections
between the constituent subunits (see Notes 7 and 8). The
Mass Spectrometry-Based Protein Modelling
227