Single-Particle Cryo-EM of Protein Complexes

Proteins were diluted to approximately 0.01–0.02 mg/mL with 10 mM HEPES, pH 7.0, 150 mM NaCl, adsorbed to a freshly glow-discharged carbon-coated grid, washed with the same buffer, and stained with 0.7% uranyl formate. Datasets were collected at a magnification of 100,000 using SerialEM (80 (link)) on an FEI Tecnai T20 microscope equipped with a 2k x 2k Eagle CCD camera and operated at 200 kV. The nominal magnification was 100,000 and the pixel size was 0.22 nm. Particles were selected from micrographs automatically using in-house written software (YT, unpublished), followed by manual correction using EMAN2 (81 (link)), when necessary. Reference-free 2D classifications were performed with Relion 1.4 (82 (link)). Fractions of prefusion and postfusion molecules were determined by calculating the numbers of particles that contributed to prefusion and postfusion classes.