Talos arctica transmission electron microscope

Aliquots of 3 μL of purified protein were applied to glow-discharged 300-mesh Au R1.2/1.3 Holey carbon grids (Quantifoil). For the peptide-bound form, 1 mM 9-mer peptide (¹RRYQKSTEL⁹) was added to DM/CHS-purified ΔTMD0 and incubated on ice for 30 min. For the ADP·BeF₃-bound form, the protein reconstituted into the nanodiscs formed by E. coli polar lipids was incubated with 1 mM 9-mer peptide, 2 mM ATP, 2 mM MgCl₂, 8 mM NaF, and 2 mM BeSO₄ at 37 °C for 10 min. The grids were blotted for 1–2 s with a blot force of 3–5 and plunge-frozen in liquid ethane cooled with liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific). All cryo-EM data were collected using a 200 kV Talos Arctica transmission electron microscope (Thermo Fisher Scientific) equipped with a K3 detector and BioQuantum energy filter (Gatan). All micrograph stacks were collected using EPU software in counting mode with a pixel size of 0.83 Å/pixel, a defocus range of −0.8 to −2.2 μm, and a dose rate 13.3 e⁻/Ų/s (total dose 40 e⁻/Ų, 50 frames).

The proper eucentric height of the specimen was determined using Leginon immediately before starting data collection. Parallel illumination of the beam was achieved earlier in the previous week by first adjusting the defocus to bring the objective aperture into focus in the front focal plane of the diffraction lens in diffraction mode followed by adjustments of the beam intensity to minimize the spread of diffraction. Data were acquired using the Leginon automated data-acquisition program (Suloway et al., 2005 ▸ ). Image pre-processing [frame alignment with MotionCor2 (Zheng et al., 2017 ▸ ) and CTF estimation using CTFFIND4 (Rohou & Grigorieff, 2015 ▸ )] were performed using the Appion processing environment (Lander et al., 2009 ▸ ) for real-time feedback during data collection. Images were collected on a Talos Arctica transmission electron microscope (Thermo Fisher) operating at 200 kV with a gun lens of 6, a spot size of 6, a 70 µm C2 aperture and a 100 µm objective aperture using beam-image shift. Movies were collected using a K2 direct electron detector (Gatan) operating in counting mode at 45 000×, corresponding to a physical pixel size of 0.91 Å per pixel, with a 10 s exposure using 200 ms per frame. Using an exposure rate of 4.204 e per pixel per second, each movie had a total dose of approximately 42 e Å⁻² for the 2111 movies over a defocus of 0.8–2 µm.

For cryo-EM, sample vitrification was carried out using a Mark IV Vitrobot (Thermo Fisher Scientific). 3 μl hnRNPDL-2 amyloid fibrils diluted in MQ water at a final concentration of 0.25 mg/mL were applied to a C-Flat 1.2/1.3-3Cu-T50 grid (Protochips) previously glow-discharged at 30 mA for 30 s in a GloQube (Quorum Technologies). Sample was incubated on grid for 60 s at 4 °C and 100% humidity, blotted and plunge-frozen into liquid ethane. Vitrified samples were transferred to a Talos Arctica transmission electron microscope (Thermo Fisher Scientific) operated at 200 kV and equipped with a Falcon 3 direct electron detector (Thermo Fisher Scientific) and EPU 2.8 (Thermo Fisher Scientific) software. A total of 1114 movies were collected using EPU 2.8 (Thermo Fisher Scientific) in electron counting mode with an applied dose of 40 e^-/Ų divided in 40 frames at a magnification of 120 kx. All the micrographs were acquired with a pixel size of 0.889 Å/pixel and a defocus range of −1.0 to −2.2 μm.