Cryo-electron microscopy (cryo-EM) is capable of capturing such polymer structures without having to break them down and crystallize single subunits for x-ray diffraction analysis. X?ray diffraction might allow higher resolution, but it would lose all information concerning contacts between the subunits during polymerization. "With cryo-EM, by changing the parameters we could see how different polymer forms convert," says Nogales.
Nogales and Wang first tackled the depolymerization process, watching what happens to microtubules when GDP is bound to tubulin. During this process tubulin protofilaments curve back sharply from the end of the microtubule, forming "peels," like a wood shaving curling up from a plane. Wang found conditions for which the curls of GDP-bound tubulin close into tubes, with the protofilaments tightly wrapped around the tube axis. These tubes are double-layered, a property that contributes to their stability -- but it also put the brakes on the project for a long time.
"The key was to get the tubulin assemblies locked at the correct states suitable for structural analysis, but even more challenging was finding an algorithm to reconstruct the double-layered GDP-tubulin tubes," says Wang.
"Due to the double-layered property, it was impossible for us to use classical helical reconstruction methods," he says, "but there were no other methods available when we started the project. We had to spend over two years developing and implementing our own algorithm. Once it was ready, we were able to get the correct reconstruction in less than three months."
Next the researchers tackled the polymerization process, watching what happens when GTP is
Source:DOE/Lawrence Berkeley National Laboratory