title: Structure of γ-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure.
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.
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA, USA. Electronic address: cgreen@salilab.org.
authors.forename: Charles H
authors.initials: CH
authors.lastname: Greenberg
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Kollman
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Zelter
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Johnson
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address: tdavis@uw.edu.
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA; Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA, USA. Electronic address: agard@msg.ucsf.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Agard
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA, USA. Electronic address: sali@salilab.org.
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D012984:Software; D040681:Structural Homology, Protein; D014404:Tubulin / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D014404:Tubulin
keywords: Allosteric regulation; Comparative models; Computational structure prediction; Integrative modeling; Microtubule nucleation