Prior to preparing grids, RecBCD was thawed and desalted into 20 mM Tris-HCl (pH 8.0), 50mM NaCl, 0.5 mM TCEP using Sephadex G25 spintrap columns (GE Healthcare). The protein was mixed with a 1.5 fold excess of DNA substrate for 10 min at room temperature. Final concentrations were 1 μM RecBCD and 1.5 μM DNA substrate. A RecBCD-Chi complex was prepared using a Chi-containing DNA substrate with a Y spacing of 8 and a Z spacing of 4 (see Figure 1 ). For the Chi-minus dataset, a complex was prepared using the negative control substrate lacking Chi. For the Chi-minus 2 dataset, a complex was prepared using a Chi-containing DNA substrate with a Y spacing of 6 and a Z spacing of 4 (Figure 1 ). For the Chi-plus 2 dataset, a complex was prepared using a Chi-containing DNA substrate with a Y spacing of 10 and a Z spacing of 4 (Figure 1 ). Quantifoil R2/2 μm holey carbon film grids (300 mesh) were treated by plasma cleaning for 30 s before being covered with graphene oxide sheets. In order to improve grid preparation reproducibility and hydrophilicity of the graphene oxide, the following method was used: 5μl of graphene oxide solution (Aldrich 763705, 2mg/ml) were mixed with 5μl of 1.5% w/v solution of nonionic detergent n-Dodecyl-β-D-Maltoside. The mixture was diluted 100 times with water and 5ul was applied to the carbon side of the grid. A sharp edge of a piece of filter paper was applied to the centre of the opposite surface of the grid to pull the graphene oxide solution through the grid. Grids were used within 30 minutes of graphene oxide application. Sample (4 μL) was evenly applied to the graphene oxide-coated side of the grid, followed by a 5 s wait time, 1 s blot time, and freezing in liquid ethane using a Vitrobot Mark IV (FEI). The Vitrobot chamber was maintained at close to 100% humidity at 4°C.
The dataset with the Chi substrate was collected using a Titan Krios microscope operated at 300 KV at eBIC, Diamond, UK. Zero loss energy images were collected automatically using EPU (FEI) on a Gatan K2-Summit detector in counting mode with a pixel size of 1.047 Å. A total of 3,721 images were collected with a nominal defocus range of −1.3 to −2.5 μm in 0.3 μm increments. Each image consisted of a movie stack of 40 frames with a total dose of 45 e-/Å2 over 10 s corresponding to a dose rate of 5 e-/pixel/s. The dataset with the Chi-minus control substrate was collected using a similar collection strategy again with a Gatan K2-Summit detector and Titan Krios microscope at eBIC, Diamond, UK. The pixel size was 1.048 Å and a total of 788 images were collected with a similar defocus range to the above. A total dose of 45 e-/Å2 was split into 40 frames over 7 s, corresponding to a dose rate of 7 e-/pixel/s.
The datasets with the Chi-minus 2 and Chi-plus 2 were collected using a Titan Krios microscope operated at 300 KV at eBIC, Diamond, UK. Zero loss energy images were collected automatically using EPU (FEI) on a Falcon3 detector in integrating mode with a pixel size of 1.085 Å. A total of 4,013 images (Chi-minus 2 substrate) and 3,613 images (Chi-plus 2 substrate) were collected with a nominal defocus range of −1.2 to −2.7 μm in 0.3 μm increments. Each image consisted of a movie stack of 39 frames with a total dose of 76 e-/Å2 over 1 s corresponding to a dose rate of 89 e-/pixel/s.
The dataset with the Chi substrate was collected using a Titan Krios microscope operated at 300 KV at eBIC, Diamond, UK. Zero loss energy images were collected automatically using EPU (FEI) on a Gatan K2-Summit detector in counting mode with a pixel size of 1.047 Å. A total of 3,721 images were collected with a nominal defocus range of −1.3 to −2.5 μm in 0.3 μm increments. Each image consisted of a movie stack of 40 frames with a total dose of 45 e-/Å2 over 10 s corresponding to a dose rate of 5 e-/pixel/s. The dataset with the Chi-minus control substrate was collected using a similar collection strategy again with a Gatan K2-Summit detector and Titan Krios microscope at eBIC, Diamond, UK. The pixel size was 1.048 Å and a total of 788 images were collected with a similar defocus range to the above. A total dose of 45 e-/Å2 was split into 40 frames over 7 s, corresponding to a dose rate of 7 e-/pixel/s.
The datasets with the Chi-minus 2 and Chi-plus 2 were collected using a Titan Krios microscope operated at 300 KV at eBIC, Diamond, UK. Zero loss energy images were collected automatically using EPU (FEI) on a Falcon3 detector in integrating mode with a pixel size of 1.085 Å. A total of 4,013 images (Chi-minus 2 substrate) and 3,613 images (Chi-plus 2 substrate) were collected with a nominal defocus range of −1.2 to −2.7 μm in 0.3 μm increments. Each image consisted of a movie stack of 39 frames with a total dose of 76 e-/Å2 over 1 s corresponding to a dose rate of 89 e-/pixel/s.
