Plunge-Freezing of Lysenin Oligomers on Graphene Oxide

Specimens were plunge-frozen using a custom fabricated plunger at 4 °C. Lysenin oligomers (3 μl of ∼0.2 mg ml⁻¹) were applied to copper 300 square mesh Quantifoil R1.2/1.3 holey-carbon grids (Quantifoil Micro Tools, GmbH) overlaid with graphene oxide (see below) and left to adhere for 30 s. The grids were then blotted from the specimen side for 10 s before being plunge-frozen in liquid ethane. Specimens were imaged on an FEI Titan Krios transmission electron microscope operating at an accelerating voltage of 300 kV. Micrographs were recorded in super-resolution counting mode using a Gatan K2 Summit direct electron detector at the end of a Gatan Quantum energy filter in zero-loss mode and an energy selecting slit width of 20 eV. The total dose on the specimen was 47 e⁻ per Ų fractionated over 20 frames with a calibrated pixel size of 0.715 Å for the super-resolution micrographs.