Structure of γ-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure

• DOI: 10.1016/j.jsb.2016.03.006
• Publication Date: 2016-03-08T20:16:20Z
• Authors: Charles H. Greenberg; Justin Kollman; Alex Zelter; Richard Johnson; Michael J. MacCoss; Trisha N. Davis; David A. Agard; Andrej Sali
• Keywords: Cryo-Electron Microscopy; Sequence (biology)

Modeling protein complex structures based on distantly related homologues can be challenging due to poor sequence and structure conservation. Therefore, utilizing even low-resolution experimental data can significantly increase model precision and accuracy. Here, we present models of the two key functional states of the yeast γ-tubulin small complex (γTuSC): one for the low-activity "open" state and another for the higher-activity "closed" state. Both models were computed based on remotely related template structures and cryo-EM density maps at 6.9Å and 8.0Å resolution, respectively. For each state, extensive sampling of alignments and conformations was guided by the fit to the corresponding cryo-EM density map. The resulting good-scoring models formed a tightly clustered ensemble of conformations in most regions. We found significant structural differences between the two states, primarily in the γ-tubulin subunit regions where the microtubule binds. We also report a set of chemical cross-links that were found to be consistent with equilibrium between the open and closed states. The protocols developed here have been incorporated into our open-source Integrative Modeling Platform (IMP) software package (http://integrativemodeling.org), and can therefore be applied to many other systems.