Rings of guanosine diphosphate (GDP)-tubulin formed in the presence of divalent cations have been studied using conventional negative stain and cryo-electron microscopy. The structure of such rings resembles that of depolymerizing microtubule ends and corresponds to an “unconstrained” conformation of tubulin in its GDP state. The use of cryo-techniques has allowed us to image the ring polymers free from dehydration and flattening artifacts. Preparations of frozenhydrated GDP-tubulin rings are generally heterogeneous and contain a mixture of double, triple, and incomplete rings, as well as spirals and some rare single rings. Images of different polymer types can be identified and classified into groups that are then amenable for averaging and single particle reconstruction methods. Identifying the differences in tubulin structure, between straight and curve protofilaments, will be important to understand the molecular bases of dynamic instability in microtubules.
- cryo-electron microscopy
- single particles