Ultrascan 1000 xp ccd

To examine actin bundling, 500 nM MBP-Hof1-FL-6His was mixed with 1 µM preformed F-actin filaments. Then, 3 µl of protein complexes were applied to the carbon-coated glow-discharged copper grids. Samples were blotted and negatively stained with 1.0% uranyl acetate two times for 30 s each. To examine Hof1–septin interactions, 500 nM MBP-Hof1-FL-6His was mixed with 50 nM of septin rods and applied to glow-discharged grids and negatively stained as above. Air-dried grids were imaged on a JEOL 2100 transmission electron microscope equipped with a Gatan Ultrascan 1000XP CCD camera and operated at 200 kV. The data were collected under low-dose conditions at a ∼1.5 μm defocus and 40,000 magnification resulting in 2.5 A pixel size. Class averages of Hof1 particles on actin bundles and on the carbon substrate shown in Figure 3 were collected in EMAN2 (Tang et al., 2007 (link)). A total of 50 particles for each set were cut from the micrographs and aligned against one another. The total sums of bound Hof1 and free Hof1 have comparable size and shape.