ET class averaging of individual microtubules reveals novel densities.

(A) 628 particles containing microtubules and paddlewheels were picked from individual ET tilt series of 4 centrioles. Class-averaging resulted in 12 classes, 3 of which were well defined and together contained 92% of input particles (classes 1–3, colorized with the LUT “Fire”, low intensities in blue, high intensities in magenta and red). (B) Line intensity profile of classes 1–3 along the lines indicated in (A) (10 pixels wide). The microtubule displayed consistently more density on the side located under the paddlewheel (“A”) than on the other side (“B”). (C) Overlay of U-Ex-STED images for FLAG::SAS-1, SPD-5 and microtubules, together with EM images (inverted grey levels) of centrioles from early meiotic prophase. Circularized images (left two panels), corresponding 9-fold symmetrized versions (next two panels), and magnification of the insets highlighted by the white box (very right). EM, electron microscopy; ET, electron tomography; U-Ex-STED, Ultrastructure Expansion coupled with STimulated Emission Depletion. (PDF)