R1.2 1 3 holey carbon em grids

Human Hgb was commercially sourced (Sigma-Aldrich, St Louis, MO, USA) and dissolved in 50 mM Tris buffer at pH 7.6 to a final concentration of 1.5 mg ml⁻¹. Frozen-hydrated specimens were prepared on plasma-cleaned Quantifoil R1.2/1.3 holey carbon EM grids (Quantifoil, Großlöbichau, Germany) using a Vitrobot Mark III (FEI, Hillsboro, OR, USA) 5 s blotting time, 85% humidity and −5 mm blotting offset.

NanR-dimer₁/DNA hetero-complex, was prepared by mixing NanR (17 µM) and (GGTATA)₂-repeat DNA (8.5 µM). NanR-dimer₃/DNA hetero-complex was prepared by mixing NanR (85 µM) and the (GGTATA)₃-repeat operator DNA (8.5 µM). To reduce sample heterogeneity and remove aggregates, each hetero-complex was purified using size-exclusion chromatography (Supplementary Figs. 8a and 10a, respectively). Following equilibration for 1 h at 4 °C, the sample was loaded onto a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with buffer A. Fractions consistent with hetero-complex formation were pooled and diluted to a final concentration of 0.5 mg mL⁻¹. Frozen-hydrated samples were prepared on plasma-cleaned Quantifoil R1.2/1.3 holey carbon EM grids (Quantifoil) using a Vitrobot Mark IV vitrification robot (FEI) with a 3-s blotting time, 100% humidity, and −3 mm blotting offset.