Superose 6 increase 3.2 300

Purified CPF complexes were made by mixing 5 μM polymerase module, 5 μM Ysh1-Cft2-phosphatase module and 15 μM Mpe1 or any variants thereof. The final volume was brought to 50 μl with SEC 150 KCl buffer (20 mM HEPES pH 8, 150 mM KCl, 0.5 mM Mg(OAc)₂, 0.5 mM TCEP). Sample was kept on ice before being loaded on a Superose 6 Increase 3.2/300 (Cytiva, cat. No. 29091598) equilibrated in SEC 150 KCl buffer. For polymerase module complexes to be used in EMSAs or polyadenylation assays, 10 μM of polymerase module was mixed with 30 μM Mpe1 or variants thereof and purified using a Superose 6 Increase 3.2/300 column with SEC 50 buffer (20 mM HEPES pH 8, 50 mM NaCl, 0.5 mM TCEP). Fractions showing all components of the respectively assembled complexes with correct stoichiometry were pooled, concentrated in an Amicon concentrator (100 kDa cutoff) and flash frozen. Concentration was determined using the theoretical extinction coefficient for polymerase module-Mpe1 (330,960 M^-1 cm^-1), CPF^ΔMpe1 (632,930 M^-1 cm^-1) or CPF (665,360 M^-1 cm^-1) calculated in ProtParam. For cryo-EM sample preparation, 3 μl from the peak fraction were used per grid.

Purified Spike and VHH Re5D06 (produced in P. pastoris, see above) were mixed in a 1:9 molar ratio (RBD‐to‐nanobody ratio of 1:3) and subjected to size exclusion chromatography (Superose 6 Increase 3.2/300, Cytiva; equilibrated with 2 mM Tris/HCl pH 8.0, 150 mM NaCl). 3 μl of the Spike⋅Re5D06 peak fraction (1 mg/ml; see Appendix Fig S8A) was applied to freshly (air) glow‐discharged Quantifoil UltrAuFoil R2/2 grids (200 mesh). The grids were blotted at 4°C and 100% humidity for 3–6 s with a blot force of 5 and plunge‐frozen in liquid ethane using a Vitrobot Mark IV (FEI Company). Grids were screened on a Glacios 200 kV TEM (Thermo Fisher Scientific) equipped with a Falcon III detector (Thermo Fisher Scientific). A blotting time of 4 s was optimal in this setup and yielded a gradient of ice thickness across the holes (Appendix Fig S8B), which was important for reaching good angular particle distribution coverage in the 3D reconstructions (Appendix Fig S8C and D, S10 and S11).