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Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50 Page 9 of 16

Figure 2. Comparison of different TP901-1 CI-NTD structures. (A) Aromatic residues Tyr3, Tyr5, Trp43, and Phe67 of TP901-1 Mor
form a cavity in which Gln55 from CI must fit for complex formation; (B) the conformation of Glu69 in the structures of CI without Mor
would cause severe clashes with Phe67 of Mor in the complex; (C) the structures of the C-terminal region from Asp76 to Val80 are
similar in CI-NTD alone (5A7L, 8QAO from this manuscript) and in the complex with Mor (6TRI). The CI-NTD:Mor complex structure
is shown in light green (CI) and teal (Mor) cartoon representation with green sticks for highlighted side chains. The shown sticks are
from CI-NTD containing structures 3ZHI (light blue), 3ZHM (yellow), 5A7L (grey), and 8QAO from this manuscript (salmon). CI-NTD:
N-terminal domain of Phage repressor.

modeled as Ala [Supplementary Figure 1] clearly indicates that the Glu69 side chain density would clash
with Phe67 of Mor in the complex; thus, a considerable rearrangement is required on complex formation.
In the Mor NMR structure ensemble (PDB code 6TOE), the Phe67 side chain can be in alternate
conformations, all clashing with Glu69 in the uncomplexed CI-NTD crystal structures.

The extreme C-termini of the CI-NTDs are difficult to compare as the sequences differ here due to the
presence of tags and different construct lengths. However, we note that the C-terminal region Asp76-Val80
is very similar in the CI:Mor complex and in the only two other structures that contain this region
[Figure 2C]. In the most recent structure, the C-terminus folds against the rest of the protein beyond Val80
and becomes difficult to trace, but this is unlikely to be the case in the full-length protein, as this region is
the linker to the dimerization region.