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Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50 Page 7 of 16

Table 2. Crystallographic statistics of the TP901-1 CI-NTD crystal structure

Beamline, date of data collection ID23-2, ESRF, Grenoble; 21/01/2022
Auto-processed dataset used XDSAPP
Wavelength [Å] 0.8731
Space group P 4 2 2
3
1
No. of molecules/asymmetric unit 1
Cell parameters
(a, b, c) [Å] 48.4, 48.4, 87.0
(α, β, γ) [°] 90.0, 90.0, 90.0
Resolution [Å] 43.48 - 1.29
(1.37 - 1.29)*
Completeness [%] 94.0 (69.5)
R [%] 25.6 (375.0)
meas
R pim [%] 5.1 (198.4)
I/σ(I) 13.71 (0.48)
CC [%] 99.9 (21.7)
1/2
Observed reflections 891,991
Unique reflections 46,858
Redundancy 19.04
R [%] 14.97
work
R free [%] 18.29
RMSD
Bond lengths [Å] 0.0179
Bond Angles [°] 2.4631
#
Ramachandran statistics (%)
Favored 98.7
Allowed 1.3
Outlier 0
# [34]
*The values in parentheses are for the highest resolution shell; Ramachandran statistics are calculated in RAMPAGE . CI-NTD: N-terminal
domain of Phage repressor.

Isoelectric focusing
Protein-protein interactions between φ13 CI and Mor were assessed using isoelectric focusing (IEF)
electrophoresis under native conditions, similarly as previously for variants of TP901-1 CIΔ58 (a truncated
version of CI missing the last 58 residues) and Mor . Samples were run on a SERVALYT™ PRECOTES™
[22]
Wide Range pH 3-10 gel with SERVA IEF Marker 3-10, Liquid Mix as a protein standard. Samples were
diluted to 12.7 M, and for the complex samples, CI and Mor were mixed in different ratios and incubated at
4 °C for 60 min. Samples were loaded using an applicator strip with volumes ranging from 5-15 µL in order
to get comparable amounts of each protein in the different wells. The gel was run using a Multiphor II EIF
chamber (Amersham Biosciences) with running conditions of 2,000 V, 12 mA, and 24 W for 180 min, and
the gel was cooled to 5 °C by a thermostatic circulator. For protein detection, the gel was first fixed using
20% (w/v) trichloroacetic acid for 20 min, followed by a rinse using 3% (v/v) phosphoric acid for 5 min. The
gel was stained with SERVA Violet 17 [200 mg SERVA Violet 17 mixed with 100 mL milli-Q water and
100 mL 20% (v/v) phosphoric acid] for 30 min and subsequently destained with 3% (v/v) phosphoric acid
until no background staining was visible. The gel was scanned using a Gel Doc EZ system from Bio-Rad and
analyzed using Image Lab software.