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Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50 Page 5 of 16

METHODS
Protein expression and purification
TP901-1 CI-NTD was expressed and purified as in . Briefly, BL21(DE3) E.coli was transformed with
[24]
pET30a(+) with kanamycin resistance encoding a synthetic gene for CI-NTD (residues 1-89 with a
C-terminal His-tag, see construct list in Table 1). 10 mL lysogeny broth (LB) medium starter cultures were
grown for 16 h, and used to inoculate 1 L of LB medium expression cultures (50 g/mL kanamycin). A final
concentration of 1 mM isopropylthio-β-galactoside was added when the culture had reached an optical
density (OD) of 0.5-0.6. Growth was continued overnight at 25 °C prior to harvesting the soluble fraction
after sonication in lysis buffer (20 mM imidazole, 20 mM Tris pH 7.5, 1 M NaCl).

Protein was purified on an ÄKTA Purifier system (GE HealthCare/Cytiva), first by immobilizing on a 1 mL
His-Trap™ HP (Cytiva) in lysis buffer, followed by a short wash in the same. Protein was eluted with 20 mM
Tris, 100 mM NaCl, 500 mM Imidazole, pH 7.5. A final purification step was carried out by Size-Exclusion
Chromatography on a HiLoad 26/60 Superdex 200 column with 20 mM Tris, 100 mM NaCl pH 7.5 as
eluent.

Φ13 CI and φ13 Mor with N-terminal His tags (sequences in Supplementary Table 1) were produced as φ13
[28]
CI in , following basically the same procedure as above. All synthetic genes were from Genscript and
codon-optimized.

Protein concentration was determined using A measured with a NanoDrop ND-1000 UV/Vis
280
spectrophotometer using the estimated absorption coefficient calculated from the sequence using the
Expasy Protparam tool (https://web.expasy.org/protparam/). Protein was concentrated using Amicon Ultra
3kDa cutoff filters and stored frozen at -20 °C.

Crystal structure determination of CI-NTD
Using the sitting-drop vapor diffusion technique, a JCSG+ screen (Qiagen) was set up for the TP901-1 CI
NTD protein (buffer: 20 mM Tris, 100 mM NaCl pH 7.5). The Oryx 8 Protein Crystallization Robot
(Douglas Instruments Ltd.) was used to set up the screen at room temperature in MRC2 Well
Crystallization plates (SwissCI). The stock protein concentration used was 8.14 mg/mL. In each well, there
was a reservoir volume of 100 µL (screen solution) and sitting drops of 0.3 µL The protein:reservoir ratio
was 3:1 in the top drop and 1:1 in the bottom. For optimization, 2D grids for four chosen conditions were
also set up using sitting drop vapor diffusion at room temperature. The crystals sent to the synchrotron
were produced in the optimized condition consisting of 28% w/v PEG 4000, 0.17 M ammonium sulfate, and
15% glycerol. The protein concentration in this optimized condition was 3.3 mg/mL in 20 mM Tris pH 7.5,
100 mM NaCl (protein stock), and the protein: reservoir ratio was 3:1 in the top drop and 1:1 in the bottom,
and the drop volume was 0.4 µL. Crystals were frozen in liquid nitrogen directly in mother liquor without
the addition of cryoprotectant. X-ray diffraction data were collected at beamline ID23-2 ESRF, Grenoble,
France. One of the datasets with 1.29 Å resolution was chosen to determine the structure. The previous
TP901-1 CI-NTD structure (PDB code:3ZHI) was chosen as the reference structure to do the Molecular
replacement in the CCP4 suite (MOLREP ). Further refinement was performed alternating
[31]
[30]
[33]
computational refinement in REFMAC5 and manual rounds in COOT until reaching a final R factor
[32]
value of 14.97% and an R free value of 18.29%. Data statistics of X-ray diffraction and structure refinement
statistics are shown in Table 2.