Ultrascan 4000 4k x 4k ccd camera

Nanoparticles diluted to 0.01 mg/mL in 50 mM Tris pH 8, 150 mM NaCl, were adsorbed to glow-discharged home-made carbon-coated copper grids for 30 seconds. The excess liquid was blotted away with filter paper (Whatman 1) and 3 μL of 2% w/v uranyl formate stain were applied to the grids. Finally, the stain was blotted away, and the grids were allowed to air dry for 1 min. Grids were imaged on a 120kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k x 4k CCD camera at 67,000 nominal magnification using a defocus ranging between 1.0 and 2.0 μm and a pixel size of 1.6 Å.

Protein was adsorbed to glow-discharged carbon-coated copper grids for 1 min prior to a 3X wash with water and 2% uranyl formate staining. Micrographs were recorded using the Leginon software (Suloway et al., 2005 (link)) on a 100kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k x 4k CCD camera at 67,000 nominal magnification. The defocus ranged from 1.0 to 2.0 μm and the pixel size was 1.6 Å. Particles were picked automatically in a reference free manner using DogPicker. Contrast transfer function (CTF) estimation was performed using GCTF (Zhang, 2016 (link)). Class averages were generated using Relion2.1 (Kimanius et al., 2016 ).