Fig. 3: Negative-staining EM micrographs and cryo-EM structure of the MT assembly intermediate.

a–c EM micrographs of negatively stained specimens. Scale bar: 50 nm. a Helical ribbons appear at the growing end of GTPγS-MT at 28 °C with 2 mM Mg²⁺. b Only GTPγS-Tube exists in the condition of 10 mM Mg²⁺. c GTPγS-Tubes convert into MT directly in two different ways. Left (Conversion-1): One Tube converts into one MT. Right (Conversion-2): One Tube splits into two MTs. The branch point is pointed out by the black arrow. Three independent replications of negative staining were conducted. d–f Cryo-EM structure of GTPγS-Tube-KMD complex. d The density map of GTPγS-Tube-KMD complex. Top: Slightly tilted top view of the whole complex. Middle: Side view of the outer tubulin (medium purple) and kinesin (yellow). Bottom: Side view of the inner tubulin (hot pink) and kinesin (cyan). The dashed line indicates the direction of tubulin heterodimer arrangement. e A cross section of the whole map. Atomic models of tubulin and kinesin are fitted in the density map, and three tubulin-kinesin interfaces are labeled (inset). f The density map and corresponding models of four adjacent tubulin heterodimers in the outer layer of GTPγS-Tube. “Tube-bond” and “MT-bond” interfaces are indicated by the black box with dashed line. Secondary structures engaging in these interfaces are labeled.