Leica em gp

C. elegans katanin p60 (MEI-1) with a Walker B mutation (E293Q) at ~1mg/ml (18 μM) in 20 mM HEPES-KOH pH 7.5, 300 mM KCl, 10 mM MgCl₂, 1 mM ATP, 2 mM TCEP was mixed on ice with excess of poly(E) molecular weight 3 kDa (30 μM) (Alamanda Polymers, CAS#26247–79-0) just before cryo-EM grid preparation. 5 μl of sample were applied to a glow-discharged C-flat holey carbon on gold grids with 2 μm hole, 1 μm space (CF-2/1–4Au). The grids were blotted for 5 seconds at 90% humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen using Leica EM GP (Leica Microsystems, Germany). A dataset of 5,911 movie stacks was collected on a Titan Krios microscope (FEI) operated at 300 kV, equipped with a K2 Summit direct electron detector camera, operated in super-resolution mode, energy filter slid width of 20 eV, C2 aperture 70 μm and 100 μm objective aperture. The movies were recorded at a nominal magnification of 130,000X, corresponding to a physical pixel size of 1.08 Å/pixel at a defocus range from −1.2 μm to −2.5 μm. The dose rate was 8.5 e⁻/pix/s. 50-frame movie stacks were recorded for 10 s with a cumulative electron dose of 73 e⁻/Ų (Table S1). Data collection was automated with Leginon (Suloway et al., 2005 (link)).

Vitrified samples of GluK2 solublized in DDM-CHS were prepared with protein concentrated to 4.2 mg/mL. A volume of 3 μL was added to R1.2/1.3 holey carbon grids (Quantifoil) rendered hydrophilic with self-assembled monolayer functionalization^{27 (link)}, and grids were frozen with a Vitrobot Mk IV robot (FEI Company, Hillsboro, OR, USA) or a Leica EM GP (Leica Microsystems Inc., Buffalo Grove, IL, USA).
Data was collected using a Titan Krios, operated at 300 kV, aligned for parallel illumination, and equipped with a GIF Quantum Energy Filter (Gatan, Inc.) operated in zero-energy-loss mode with a slit width of 20 eV (Extended Data Table 2). Images were acquired manually on a K2 Summit camera (Gatan, Inc.), at 105,000 X nominal magnification corresponding to a 1.324 Å physical pixel size. Each exposure was recorded in super-resolution mode as a 38-frame movie, with dose rate and exposure time of 3 e Å⁻¹ s⁻¹ and 15 s, respectively.