Em grid plunger

Aliquots of 3.5 ul of each sample was applied to plasma-cleaned (Fishione Inc.) C-flat grids (Protochips, CF-1.2/1.3–4C), blotted on the sample side with filter paper for 2 seconds (22 °C, 90% humidity) and then plunged into liquid ethane with a Leica EM Grid Plunger (Leica Microsystems). For the dyn^GTP samples, after 3.5 ul sample was applied to the grids in the grid plunger, GTP was added and plunged into ethane after 5–10 seconds. The vitrified samples were stored in liquid nitrogen before examination by cryo-EM. For the dyn^GMPPCP polymer samples, images were recorded during three sessions on a Titan Krios microscope (FEI) at 300 kV and recorded at 22,500X magnification with a defocus range of 1.0–3.0 μm on a K2 summit camera in counting mode. For the GMPPCP treated sample containing partially constricted polymers and for the dyn^GTP sample, images were recorded on a TF20 microscope (FEI) at 200 kV and recorded at 29,000X magnification, with a defocus range of 1.5–3.0 μm on a K2 summit camera in counting mode (Extended Data Table 1).

BAM-^MBP−76EspP native nanodiscs were diluted in TN^low buffer at a concentration of ~2 –8 mg L⁻¹, and 3 μL of sample was applied onto glow-discharged C-flat grids (EMS CF-1.2/1.34Au-50) for 3 sec before plunge freezing in liquid ethane using a Leica EM Grid Plunger (Leica Microsystems). Datasets were collected at the NIH Multi-Institute Cryo-EM Facility (MICEF) using a Titan Krios G3 microscope (Thermo-Fisher) operating at 300 kV. During 4 collection sessions (Figure S5, dataset 1) micrographs were collected at a magnification of 130,000x (calibrated pixel size 0.5371 Å, nominal defocus range 0.6 to 1.8 μm, 40 frames, and 60 e^-/Ų electron exposure per movie) using a Gatan K2 Summit direct electron detection camera equipped with a Gatan Quantum LS imaging energy filter with slit width set to 20 eV. After the microscope was upgraded with a Gatan K3 camera an additional collection session (Figure S5, dataset 2) was conducted at a magnification of 105,000x (calibrated pixel size 0.4281 Å, nominal defocus range 0.6 to 1.8 μm, 23 frames, and 60 e^-/Ų electron exposure per movie). Multiple collection sessions were conducted in order to allow reconstructions of multiple intermediate substates of the complex that were present in the sample.