Cryo-EM Structural Analysis of Proteins

Proteins were diluted to approximately 0.02 mg/mL with buffer containing 10 mM HEPES, pH 7.0 and 150 mM NaCl, adsorbed to a freshly glow-discharged carbon-coated copper grid, washed with the same buffer, and stained with 0.7% uranyl formate. Datasets were collected using SerialEM (69 (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, corresponding to a 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 Particles were selected from micrographs automatically using in-house written software (YT, unpublished), followed by manual correction using EMAN2 (70 (link)), when necessary. Reference-free 2D classifications were performed with Relion 1.4 (71 (link)).