| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA2 Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri—Kansas City, Kansas City, Missouri 64110, USA
(RECEIVED September 16, 2004; FINAL REVISION December 31, 2004; ACCEPTED December 31, 2004)
Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (
)4. PhK comprises two arched octameric ()2 lobes that are oriented back-to-back with overall D2 symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 Å. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90° instead of 68°, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.
Keywords: phosphorylase kinase; cryoelectron microscopy; three-dimensional reconstruction; single particle analysis; structural analysis
Abbreviations: AFM, atomic force microscopy • EM, electron microscopy • ns, negative stain • PhK, phosphorylase kinase • SAXS, small-angle X-ray scattering • STEM, scanning transmission EM • STM, scanning tunneling microscopy • TEM, transmission EM
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041123905.
Reprint requests to: Gerald M. Carlson, Department of Biochemistry and Molecular Biology, Mail Stop 3030, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA; e-mail: gcarlson{at}kumc.edu; fax: (913) 588-7007.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. S. Priddy, E. S. Price, C. K. Johnson, and G. M. Carlson Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex Protein Sci., June 1, 2007; 16(6): 1017 - 1023. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. S. Priddy, C. R. Middaugh, and G. M. Carlson Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+ Protein Sci., March 1, 2007; 16(3): 517 - 527. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
