The genome ejection of P68 was induced by exposing it to pH 4.2, as described previously for phage phi29 (41). P68 at a concentration of 2 mg/ml in phage buffer [50 mM tris (pH 8.0), 10 mM CaCl2, and 10 mM NaCl] was mixed with liposomes at a concentration of 5 mM in phage buffer in a volume ratio of 1:10. The pH of the mixture was changed to acidic by three rounds of concentrating and diluting with 0.1 M sodium acetate, 300 mM ammonium sulfate (pH 4.2) buffer using 100-kDa Amicon ultra centrifugal filters. The phage was incubated in the acidic buffer for 1 hour. The mixture was applied to holey carbon–coated copper grids (R2/2, mesh 200; Quantifoil), blotted, and flash-frozen in liquid ethane.

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