Eagle 2k 2k ccd camera

The purified Env-C3d fusion trimers were analyzed by negative stain electron microscopy (NS-EM). A 3- µl aliquot containing ~0.01 mg/ml of the sample was applied for 15 s onto a carbon-coated 400 Cu-mesh grid that had been glow- discharged at 30 mA for 30 s, then negatively stained with 0.7% uranyl formate for 45 s. Data were collected using an FEI T20 electron microscope operating at 200 kV, with an electron dose of ~45 e^-/Ų and a magnification of 80,000 × that resulted in a pixel size of 2.74 Å at the specimen plane. Images were acquired with an Eagle 2k × 2k CCD camera (FEI) using a nominal defocus of 1000 nm and the Serial EM software (56 (link)). Particles were selected from the micrographs and extracted, and a reference-free 2D class averages were obtained using RELION 2.1.0 (57 (link)).

M13mp18 single strand DNA (New England Bio-labs) was denatured by heating at 70°C for five min and then cooled on ice for five min. Wild-type VirE2 and VirE2-TC proteins were added to the ssDNA in binding buffer (10 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) at a ratio of 1:10 (w/w) ssDNA:protein for full coverage of the DNA. The ssDNA-protein samples were incubated for 3h or overnight at 4°C to form the complex. For TEM analysis, 5 μl of the ssDNA-protein complex was placed on glow discharged carbon coated copper grids and negatively stained with 1% uranyl acetate for 50 sec. Samples were imaged in a Tecnai Spirit BioTWIN (FEI) operating at 120 kV. Images were recorded with an Eagle 2K*2K CCD camera (FEI).

Cells were grown in SD medium. Once they reached OD₆₀₀ ~ 0.5, 5 mL of a fixative solution (6% paraformaldehyde, 4% glutaraldehyde and Cacodylate buffer 0.2 M (pH = 7.4)) was added to 5 mL of media. The samples were gently rotated for 40 minutes at 30 °C, then centrifuged and washed twice in Cacodylate buffer and incubated again for an hour in the fixative solution. After washing with Cacodylate buffer and centrifugation, samples were embedded in 10% gelatin in water, fixed overnight with the fixative solution, washed in Cacodylate buffer, and then incubated overnight in 2.3 M sucrose and rapidly frozen in liquid nitrogen. Frozen ultrathin (70–90 nm) sections were cut with a diamond knife (Diatome AG, Biel, Switzerland) at –120 °C on an EM UC6 ultramicrotome (Leica Microsystems, Vienna, Austria). The sections were collected on 200-mesh Formvar-coated nickel grids. Contrasting and embedding were performed as previously described [46 (link)]. The embedded sections were observed in a Tecnai T12 electron microscope (FEI, Eindhoven, The Netherlands) operating at 120 kV. Images were recorded using an Erlangshen ES500W CCD camera (GATAN) or an Eagle 2 k × 2 k CCD camera (FEI). The quantification of mitochondrial area and cristae was performed using imageJ [47 ].