2k ccd camera

Purified 92BR SOSIP.664 trimer was incubated with a 6 molar excess of DH270.6 Fab at 4 °C for 1.5 h. A 2.5 µl aliquot containing ∼0.01 mg/ml of the Fab-92BR SOSIP.664 complex was applied for 30 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 5 mA for 2 min, followed by negative staining with 0.7% uranyl formate for 20 s. Samples were imaged using a FEI Tecnai T12 microscope operated at 120 kV and a magnification of ×52,000, yielding a pixel size of 2.13 Å at the specimen plane. Images were acquired with a Gatan 2 K CCD camera using a nominal defocus of 1500 nm at 10° tilt increments, up to 50°. The tilts provided additional particle orientations to improve the image reconstructions.

Purified SOSIP.664 trimer was incubated with a five molar excess of Fab at 4°C for 1 hour. A 3 μL aliquot containing ~0.01 mg/ml of the complex was applied for 30 s onto a carbon coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s, followed by negative staining with 2% uranyl formate for 20 s. Samples were imaged using a FEI Tecnai T12 microscope operating at 120kV, at a magnification of 52,000x, resulting in a pixel size of 2.13 Å at the specimen plane. Images were acquired with a Gatan 2K CCD camera using a nominal defocus of 1500 nm at 10° tilt increments, up to 50°. The tilts provided additional particle orientations to improve the image reconstructions.

Yeast cells were grown to mid-logarithmic growth phase, and 10 OD₆₀₀ units of cells were pelleted and fixed in 1 ml of fixative media (2.5% glutaraldehyde, 1.25% PFA, and 40 mM potassium phosphate, pH 7.0) for 20 min at room temperature. Cells were pelleted, resuspended in 1 ml fresh fixative media, and incubated on ice for 1 h. The cells were pelleted, washed twice with 0.9% NaCl, once with water, incubated with 2% KMnO₄ for 5 min at room temperature, centrifuged, and resuspended in 2% KMmO₄ for 45 min at room temperature for en-bloc staining^{48 (link)}. The cells were dehydrated using ethanol, embedded using Spurr’s resin (Electron Microscopy Sciences), and polymerized. Semi- and ultrathin sections were produced with a diamond knife (DiATO ME) on an ultra-microtome (Ultracut UCT; Leica Biosystems), collected on 200 mesh copper grids (Electron Microscopy Sciences), poststained with uranyl acetate and lead citrate, and visualized with a Tecnai T12 transmission electron microscope (FEI), operating at 120 kV. Pictures were recorded on a bottom-mounted 2k Å~ 2k CCD camera (Gatan). Brightness and contrast were adjusted to the entire images using Photoshop (version CC 2014).