Mark 4

Cryo-EM grids were frozen using a Vitrobot Mark IV (FEI) as follows: 3 μl of the concentrated sample was applied to a glow-discharged Quantifoil R1.2/1.3 holey carbon 400 mesh gold grid, blotted for 3–4 s in >90% humidity at room temperature, and plunge frozen in liquid ethane cooled by liquid nitrogen.
Cryo-EM data were recorded on a Titan Krios (FEI) operated at 300 kV, equipped with a Gatan K2 Summit camera. SerialEM^{39 (link)} was used for automated data collection. Movies were collected at a nominal magnification of 29,000× in super-resolution mode resulting in a calibrated pixel size of 0.51 Å/pixel, with a defocus range of approximately −1.0 to −3.0 μm. Fifty frames were recorded over 10 s of exposure at a dose rate of 1.22 electrons per Ų per frame.
Movie frames were aligned and binned over 2 × 2 pixels using MotionCor2^{40 (link)} implemented in Relion 3.0^{41 (link)}, and the contrast transfer function parameters for each motion-corrected image were estimated using CTFFIND4^{42 (link)}.

Three µl of wt PilQ sample (concentration = 1.5 mg/ml) was applied to freshly glow-discharged (at 15 mA for 25 s) Quantifoil R1.3/1.2 holey carbon grids (Quantifoil Micro Tools, Germany) back-coated with a thin carbon layer. The grids were vitrified in an FEI Vitrobot Mark IV plunge-freezer at 70% humidity and 10° C after blotting for 8–10 s. Cryo-EM images were collected in a JEOL 3200 FSC electron microscope operating at 300 kV, after coma free alignment, equipped with an in-column energy filter at a slit width of 18 eV. Images were recorded manually at a nominal magnification of 20,000x, yielding a calibrated pixel size of 1.63 Å, on a K2 direct electron detector (Gatan) operating in counting mode. Dose-fractionated 9 s movies of 45 frames were recorded with an electron dose of 0.75 e^-/Ų/frame at a defocus of 1.2–3.4 μm.

To prepare complexes for cryo-EM study, the ntDNA, tDNA and RNA oligonucletides were mixed in 2:2:1 molar ratio and annealed in a PCR thermocycler (2 min at 95 °C, 2 min at 75 °C, 5 min at 45 °C, −2 °C/min over 20 min, keep at 4 °C) in reconstitution buffer (10 mM Tris-HCl pH 8.0, 40 mM KCl, 5 mM MgCl₂). The RNAP-NusA-NusG complex was formed by mixing RNAP, nucleic acid scaffold, NusG and NusA in 1:2:3:3 molar ratio in EM buffer (10 mM HEPES pH 8.0, 150 mM KOAc, 2 mM DTT, 10 μM ZnCl₂, 5 mM Mg(OAc)₂), incubated at 37 °C for 5–10 min. For the other two complexes, RNAP-NusA and RNAP-NusG, the components were mixed with the same molar ratios. Each complex was purified by gel filtration (Superose 6 10/300 GL). The complex was then concentrated to 7-10 mg/ml using Amicon Ultra centrifugal filter units. CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)−2-hydroxy-1-propanesulfonat) was added to the sample at 8 mM final concentration just before grid freezing. C-flat grids (CF-1.2/1.3 400 mesh holey carbon) were glow-discharged with ELMO^TM glow discharge system (Cordouan Technologies) for 30 s at 2.5 mA. 4 μl of samples were applied on the grids and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI) with 95% chamber humidity at 10 °C (blot force 6, blotting time 2 s).

3 µl FAS solution (0.3 mg ml⁻¹), incubated with 1 mM NADPH and 1 mM malonyl-CoA for 5 min at room temperature, was applied to the graphene-coated Quantifoil grids. The grids were vitrified in a Vitrobot Mark IV plunge-freezer at 100% humidity and 10°C after blotting for 6–8 s. CryoEM images were collected with a Titan Krios (FEI Company, Hillsboro, Oregon, USA) electron microscope operating at 300 kV. Images were recorded automatically with EPU at a pixel size of 0.833 Å on a Falcon III EC direct electron detector (FEI Company, Hillsboro, Oregon, USA) operating in counting mode. A total of 792 dose-fractionated movies were recorded with a cumulative dose of ∼32 e⁻ Å⁻². Image drift correction and dose weighting were performed using MotionCor2 (Zheng et al., 2017 ▸ ) within the RELION-3 pipeline (Zivanov et al., 2018 ▸ ). CTF determination was performed with CTFFIND 4.1.13 (Rohou & Grigorieff, 2015 ▸ ). From a data set of 19 981 particles picked automatically with crYOLO (Wagner et al., 2019 ▸ ), 15 320 remained after 2D and 3D classification in cryoSPARC (Punjani et al., 2017 ▸ ). The particles contributing to the best 3D class were subjected to homogeneous and non-uniform refinement in cryoSPARC, yielding a map at 3.1 Å resolution, as determined by the post-processing procedure in RELION (Chen et al., 2013 ▸ ).