Temcam f416 4k ccd camera

Negatively stained samples were prepared using a solution of 2% uranyl acetate, at pH 4 [37 (link)]. A 5.0 μl aliquot was pipetted onto a negatively glow discharged continuous carbon grid (30 s at 25 mA in an Emitech K100X glow discharge system (Quorum Technologies Ltd)) [37 (link)]. The samples were incubated for one minute, washed with 3×20 μl of distilled H₂O, blotted on the edge of the grid with a filter paper, contrasted for 45 s with 5 μl of staining solution, and then blotted dry on the edge of the grid with filter paper. Each sample was imaged with an FEI Tecnai 12 electron microscope (acceleration voltage: 120 kV, C_S=6.3 mm; nominal magnification: 71, 540x; nominal underfocus: 0.5-1.5 μm) on a TVIPS TemCam-F416 4K CCD camera. Images of IM30 mutants were further processed by iterative MSA and MRA cycles. The CTF of the images was estimated in CTFFIND3 [38 (link)], and then corrected in IMAGIC-5 [39 (link)], followed by band-pass filtering and normalization in IMAGIC-5. Images characterizing the top-view were extracted as class averages, and separately refined in MSA-MRA cycles. To enhance SNR and structural features, the class averages were also 2-D symmetrized according to their ring size.