Assembly and three-dimensional image reconstruction of tubulin hoops.
The three-dimensional structure of tubulin hoops has been determined by image reconstruction. The surface lattice of hoops is similar to that of microtubules, but in addition hoops possess a superstructure of protofilament triplets. The protofilaments differ mainly in their apparent volumes and lateral spacings. The volumes depend strongly on the orientation on the carbon support, while the spacings do not. The differences of appearance do not reflect changes of intrinsic subunit structure. They are explained by differential staining related to the orientation and packing of protofilament. Microtubule-associated proteins do not contribute to the average subunit structure. All apparent protofilament structures differ from that expected from X-ray patterns of microtubules in terms of subunit tilt and distribution of contrast. It is concluded that the negatively stained structure is a reliable representation of the arrangement of protein subunits, but not of their shape. Tubulin hoops occur in conditions of microtubule assembly near the critical concentration in a stabilizing buffer. Their formation depends on microtubule-associated proteins and on the initial presence of tubulin oligomers, which may associate into short protofilament triplets. If their elongation is rapid compared to lateral aggregation, they form closed hoops. The growth phase is followed by a redistribution phase, during which hoops disappear in favour of microtubules. This behaviour is explained by kinetic overshoot assembly. Each triplet resembles an incomplete microtubule wall so that the junction between two triplets may be compared to a junction between microtubule walls. Such junctions are formed by a closely spaced pair of protofilaments. They are analogous to junctions between microtubules and incomplete microtubule walls, and they have the same clockwise curvature when viewed at the growing end.
D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D011487:Protein Conformation; D014404:Tubulin
D046911:Macromolecular Substances; D014404:Tubulin