title: Atomic models by cryo-EM and site-directed spin labeling: application to the N-terminal region of Hsp16.5.
abstract: We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1-32 were found to be disordered. The cryo-EM reconstruction at 13 A resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex.
authors.affiliation: Department of Molecular Physiology and Biophysics, Center for Structural Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
authors.forename: Hanane A
authors.initials: HA
authors.lastname: Koteiche
authors.forename: Steve
authors.initials: S
authors.lastname: Chiu
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Majdoch
authors.forename: Phoebe L
authors.initials: PL
authors.lastname: Stewart
authors.forename: Hassane S
authors.initials: HS
authors.lastname: Mchaourab
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020285:Cryoelectron Microscopy; D004578:Electron Spin Resonance Spectroscopy; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D009154:Mutation; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000378:metabolism; D013113:Spin Labels
chemical_list: D019843:Archaeal Proteins; C113795:HSP16.5 protein, Methanococcus jannaschii; D006360:Heat-Shock Proteins; D012997:Solvents; D013113:Spin Labels