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

J Struct Biol. 2016 Jun;194(3):303-10. doi: 10.1016/j.jsb.2016.03.006. Epub 2016 Mar 8.

Abstract

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.

Keywords: Allosteric regulation; Comparative models; Computational structure prediction; Integrative modeling; Microtubule nucleation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cryoelectron Microscopy / methods
  • Fungal Proteins / chemistry*
  • Models, Molecular
  • Protein Conformation
  • Software
  • Structural Homology, Protein
  • Tubulin / chemistry*

Substances

  • Fungal Proteins
  • Tubulin