Cryo-EM sample preparation, data acquisition, and initial data processing
This protocol is extracted from research article:
Structure and genome ejection mechanism of Staphylococcus aureus phage P68
Sci Adv, Oct 16, 2019; DOI: 10.1126/sciadv.aaw7414

Solution containing the phage (3.8 μl) at a concentration of 2 mg/ml was pipetted onto holey carbon–coated copper grids (R2/2, mesh 200; Quantifoil), blotted, and vitrified by plunging into liquid ethane using FEI Vitrobot Mark IV. The vitrified sample was transferred to an FEI Titan Krios electron microscope operated under cryogenic conditions and at an acceleration voltage of 300 kV. The illuminating beam was aligned for parallel illumination in NanoProbe mode, and coma-free alignments were performed to remove residual beam tilt. Imaging was done under low-dose conditions with a total dose of 21 e2 s−1. Data were collected with underfocus values ranging from −1.0 to −3.0 μm. Data acquisition was performed at a nominal magnification of ×75,000, resulting in a calibrated pixel size of 1.063 Å. Micrographs were acquired using a Falcon II direct electron detector operated in a movie mode. One-second exposure was fractionated into seven frames and saved as separate files. Automated data acquisition was done using the acquisition software EPU (FEI). The micrographs were collected in two separate sessions (1405 and 1486 micrographs) with the same microscope with identical settings. Micrographs from both sessions were merged and processed together.

The seven-frame movies were aligned globally and locally (5 × 5 patch) using the software MotionCor2 (42), and the defocus values were estimated from aligned micrographs using the program CtfFind4 (43). Power spectra of Fourier transforms of micrographs were visually inspected, and micrographs with distorted or missing Thon rings were discarded.

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