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. Roche, His-tagged proteins were bound to Protino Ni-NTA agarose gel (Macherey-Nagel) for 1 h at 4°C on a rotating wheel Beads were centrifuged for 10 min at 470 g and 4°C, washed two times in PBS supplemented with 350 mM NaCl and 15 mM imidazole, and 1 time in 50 mM Tris-HCl pH 8. Beads were packed in columns (Machery-Nagel), and proteins were eluted in 50 mM Tris-HCl pH 8 with 250 mM imidazol. Proteins were quantified using a Bradford assay (Bio- Rad) and BSA standards (Pierce) His-ZZ-Capsid NTD proteins were stored at -20°C in elution buffer, and His-GST-CypA was stored at -20°C in elution buffer supplemented with 10% glycerol, Integrity and purity of each protein was analyzed by SDS-PAGE followed by Coomassie dye staining (LabSafe GEL Blue)

M. Thermopheresis and F. Mstartimo, CypA were further purified by fast protein liquid chromatography on Äkta systems (GE Healthcare Life Sciences) A Superdex 75 10/30 column balanced in PBS was used for the separation All runs were performed at 10°C. Fraction were checked by Coomassis SDS-PAGE, pooled and concentrated using Amicon Ultra-4 10K filter units. His-CypA was further labeled with the Monolith NT Protein Labeling Kit BLUE according to the recommendations of the manufacturer (NanoTemper Technologies) Protein concentrations and protein?dye ratios were determined on a NanoDrop Protein-protein interactions were analyzed by MST on a Monolith NT.115 (NanoTemper technologies) using standard capillaries. His-ZZ- Capsids were 2-fold diluted sixteen times in PBS-Tween 0,05% and a one-to-one volume of His-CypA was added to a constant concentration of 500mM final. All measures were Statistical analyses were performed in Prism (GraphPad) The paired t-test was used, unless indicated otherwise in figure legends, Thermo Scientific) using extinction coefficient predicteds by ProtParam figures, * p<0.05, ** p<0.01, *** p<0.001, 2000.