4 k fei eagle camera

Five microlitres of purified protein was incubated on the top of freshly glow-discharged carbon-coated 200-mesh copper grids (PELCO) for 30 s at room temperature. Excess solution was removed using the Whatman filter paper. The grids were briefly rinsed three times in drops of ultrapure water and blotted after each rinse. The grids were incubated in a uranyl acetate solution (1% w/v) for 30 s. Excess stain was removed by blotting and the grids were dried at room temperature. The grids were imaged on a 120 keV Tecnai Spirit G2 microscope (FEI) equipped with a 4 K FEI Eagle camera.

Sections of 250 nm thickness were collected on palladium-copper slot grids (Science Services, Munich, Germany) coated with Formvar (Plano, Wetzlar, Germany). Protein A-gold (10 nm) was added to both sides of the sections as fiducial markers. Single axis tilt series were acquired with a FEI TECNAI F30 microscope operated at 300 kV and equipped with a 4k FEI Eagle camera over a −65° to 65° tilt range (increment 1°) and at an average defocus of −0.2 μm. Reconstruction of the tomograms and rendering of their 3D surface was performed by using the IMOD software package (version 4.9)[85 (link)] (bio3d.colorado.edu/imod).