Figure 11

Electron microscopy of the LLER protein. A. The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B. The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C. The Peak 2 fraction of the LLER protein was incubated with an anti-His₆ antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.