Tecnai g2 polara microscope

The experimental GroEL and GroEL/ES dataset were obtained in [8 (link)]. To collect the GroEL₁₄GroES₇, 1 μM GroEL₁₄ and 5 μM GroES₇ were incubated in a buffer for 15 min at 30^∘C, which contained 5mM MgCl₂, 5mM KCl, 5 mM ADP, 1mM DTT, and 12.5 mM Hepes (pH 7.5). 3.5 μl of protein solutions were confused with 0.5 μl of a 10 nm BSA-colloidal gold suspension using mesh grids. The sample was vitrified with plunge-freezing. The single-axis tilt series were obtained by a Tecnai G2 Polara microscope, which was equipped with 2k ×2k FEI CCD camera. The tilt series were acquired from tilt angle ±65^∘ with 2^∘ or 2.5^∘ angular increment at a different defocus levels between 7 and 4 μm. The object pixel size was 0.6nm.

Before making cryo-grids, the heparin-induced assembly reactions were centrifuged at 100,000 g for 30 min at 4°C. The resulting pellets were resuspended in 20 mM Tris, pH 7.4, 100 mM NaCl. Pronase-treated tau filaments (3 μl, at 2.0 mg/ml) were applied to glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh), blotted with filter paper and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV. For 2N4R filaments, imaging was performed on an FEI Tecnai G2 Polara microscope operating at 300 kV using a Falcon III detector prototype in integrating mode. A total of 717 movies of 30 frames was recorded during 1.0 s exposures, at a pixel size of 1.38 Å on the specimen, and a total dose of approximately 48 e/Ų. Defocus values ranged from −1.7 to −2.8 μm. For 2N3R filaments, imaging was performed on a Gatan K2-Summit detector in counting mode, using an FEI Titan Krios at 300 kV. A GIF-quantum energy filter (Gatan) was used with a slit width of 20 eV to remove inelastically scattered electrons. A total of 2051 movies of 44 frames was recorded during 11 s exposures, at a pixel size of 1.04 Å on the specimen, and a total dose of 50 electrons per Ų. Defocus values ranged from −0.8 to −2.2 μm. Further details are presented in Table 1.

A 3-μl aliquot of purified LV virions (∼1 mg ml⁻¹) was applied to a freshly glow-discharged 400-mesh holey carbon-coated copper grid (C-flat, CF-2/1-2C; Protochips Inc.) and blotted for 3 s, in 70% relative humidity for plunge-freezing (Vitrobot; FEI) in liquid ethane. Cryo-EM data were collected using an FEI Tecnai G2 Polara microscope (FEI) operated at 300 kV, equipped with an energy filter (GIF Quantum; Gatan, Pleasanton, CA) operating in zero-loss mode (20 eV energy selecting slit width) and a direct electron detector (K2 Summit; Gatan). Micrographs were collected with a defocus between 1.5 and 3 μm as movies (25 frames, each 0.2 s) in single electron counting mode using SerialEM52 (link) at a calibrated magnification of × 37,027, resulting in a pixel size of 1.35 Å.