2D and 3D EM visualization of the 6-11B-1 Fab within the microtubule lumen.

A-D) Microtubules polymerized from A,D) untreated B) MEC-17-treated (acetylated), or C) SIRT2-treated (deacetylated) tubulins were incubated with A-C) 6-11B-1 Fab fragments or D) GST-KHC motor domain and visualized after embedding in negative stain. The insets show expanded views of the boxed areas. White arrows in D) indicate kinesin-1 motors on the microtubule surface. Scale bars, 50 nm. E–G) Side and minus end views of 3D helical reconstructions of vitrified microtubules. Visible density thresholds have been adjusted to levels comparable to docked αβ-tubulin. All maps have been low-pass filtered to 22Å resolution. E) Control microtubule without Fab labeling. F) Cross section of acetylated microtubule decorated with 6-11B-1 Fab (orange). The structure of the αβ-tubulin dimer [30] has been docked into the right side of the density map (α-tubulin is shown in teal, β-tubulin is shown in purple). G) Cross section of deacetylated microtubule decorated with 6-11B-1 Fab (orange).