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![[Figure 2]](ih5006fig2mag.jpg)
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Figure 2
The cryo-EM processing scheme used to reconstruct doublet microtubules (DMTs). (a) Electron micrographs showing splayed axonemes at medium magnification (left) and DMTs (each marked with an arrowhead) at high magnification (right). The DMTs are picked (illustrated with a yellow line for a single DMT) prior to particle extraction. (b) Schematic showing the steps taken to reconstruct DMTs at progressively higher periodicities, starting with particles extracted every 8 nm along the longitudinal axis of the microtubule. Each step relies on mask-focused classification with masks (illustrated as yellow cylinders) covering features known to have a higher periodicity than the map to which they are applied. For example, 16 nm periodicity can be obtained by classifying on the position of FAP52 (a microtubule inner protein with 16 nm periodicity) in the 8 nm reconstruction. (c) The quality of the initial alignment can be monitored by inspecting the inner junction: a repeating pattern of FAP20 and PACRG. These proteins should be clearly distinguishable from one another in well aligned 8 nm reconstructions. |
![[Figure 2]](ih5006fig2.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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