Atomic models by cryo-EM and site-directed spin labeling: application to the N-terminal region of Hsp16.5

Structure. 2005 Aug;13(8):1165-71. doi: 10.1016/j.str.2005.05.006.


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.

Publication types

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

MeSH terms

  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Cryoelectron Microscopy
  • Electron Spin Resonance Spectroscopy
  • Heat-Shock Proteins / chemistry*
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism
  • Models, Chemical*
  • Mutation
  • Protein Structure, Tertiary
  • Solvents / metabolism
  • Spin Labels


  • Archaeal Proteins
  • HSP16.5 protein, Methanococcus jannaschii
  • Heat-Shock Proteins
  • Solvents
  • Spin Labels