K2 summit camera

For negative stain, data for mutants 5 (monomer) were collected on a Tecnai F20 microscope with a Tietz F416 CMOS detector at the New York Structural Biology Center (NYSBC). Leginon software (Suloway et al, 2005) was used for the semi‐automated collection of 825 images at a magnification of ×62,000 and a pixel size of 3 Å per pixel. For cryo‐EM data collection, 1,200 movies of mutant 5 (monomer) mixed with 2 mM AMPPNP were recorded with SerialEM (Mastronarde, 2005) at 300 kV on a Titan Krios (FEI) equipped with a K2 summit camera (Gatan) at 0.655 Å per pixel in super‐resolution mode at Janelia Research Campus. Another 664 movies of the same mutant (mutant 5—monomer) mixed with 2 mM ATP and 2 mM vanadate were recorded with SerialEM at 200 kV on a Arctica (FEI) equipped with a K2 summit camera (Gatan) at 0.578 Å per pixel in super‐resolution mode at New York University.

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)}.

In our study, we also incorporated experimental data from three different datasets obtained from the Electron Microscopy Public Image Archive (EMPIAR): EMPIAR 10,288 [14 (link)], 10,314 [15 (link)], and 10,196 [16 (link)].
EMPIAR 10,288 dataset consists of movies with 40 frames, whose size is 3838 × 3710 pixels. The pixel size is 0.86 Å and the average exposure was 1.25 $e/$ Ų. Data were acquired using a Gatan K2 SUMMIT camera on FEI Titan Krios. Gain correction images are provided.
EMPIAR 10,314 dataset includes movies with 33 frames, each having a size of 4096 × 4096 pixels. The pixel size is 1.12 Å with an average exposure of 1.51 $e/$ Ų. Data were acquired using the Falcon 3EC camera on Titan Krios. This dataset does not include gain or dark correction images.
The EMPIAR 10,196 dataset contains movies with 40 frames, each having a size of 7420 × 7676 pixels and an average exposure of 1.264 $e/$ Ų. The pixel size is 0.745 Å, which corresponds to a super-resolution setting. Data acquisition was carried out using a K2 camera on Talos Arctica. Gain correction data are provided, along with specific instructions for their application (rotation by 90 degrees left and horizontal flip).
The diversity in dataset characteristics and camera models enriched our analysis, offering valuable insights into the algorithms’ robustness and adaptability to different experimental setups.