|
Laboratory of Molecular Biophysics
|
Previous: Electron crystallography, Up: Electron Microscope Studies., Return to: Contents.
In collaboration with N. Boisset (Paris) and G. M. Carlson (Kansas).
In addition to the well established techniques of electron crystallography and helical three-dimensional reconstruction which can be applied to periodic or symmetric structures, new powerful methods for single particle analysis have been developed in the past two decades. The main difference is the way averaging is performed. Whereas, in electron crystallography or helical reconstruction, the information for several hundreds or thousands of particles is averaged directly in a Fourier transform and the reconstruction of the object is obtained by inverse Fourier or Fourier-Bessel transformation, single particle analysis works with a large number of individual images of the object and combines individual image elements. The advantage of this technique is that it is not necessary to obtain a highly regular arrangement of the object. The method has been successfully used to carry out 3-D reconstruction of large symmetric (e.g. the chaperonin ) or asymmetric macromolecular assemblies (e.g. ribosomes).
Phosphorylase kinase integrates signals from hormonal messengers and neuronal
stimuli to produce rapid activation of glycogen phosphorylase and subsequent
degradation of glycogen stores either to provide energy to sustain muscle
contraction or, in the liver, to provide other tissues such as the brain with
glucose. It is one of the most complex kinases comprising
(
ß
)4
assembly of subunits with a total
molecular weight of 1.3 x 106. The and ß
subunits are regulatory; the subunit is the catalytic subunit;
and the
subunit is identical to calmodulin and confers calcium sentivity.
The
regulation of phosphorylase kinase is described by Louise Johnson in section 4.2.
|
A 3D structure of the holoenzyme PhK has been produced at medium resolution by
electron microscopy and the random conical tilt method using the set of
programs SPIDER (1).
The 222
symmetric structure
shows a butterfly like
structure 270Å x 225Å by 160Å in overall dimensions with two wing-like lobes
connected by two oblique bridges. Comparison of the PhK model with
previous immunoelectron microscopy studies has allowed the identification of
the regulatory subunits at the tips of the lobes and the
ß regulatory
subunits at a position on the lobes closer to the
cross- bridges. Structural
studies of PhK alone and of PhK
decorated with GPb
have revealed the position
of the catalytic subunit of the phosphorylase kinase to be on the side of
the lobes close to the ends. The PhK/GPb model provides an explanation for the
formation of hybrid GPab intermediates in the PhK catalysed phosphorylation of
GPb, as previously observed by other authors.
|
We would like to pursue this structural work of Phk at higher resolution.
1. Franck, J. Three dimensional electron microscopy of macromolecular assemblies. 1996, San Diego: Academic Press.
Previous: Electron crystallography, Up: Electron Microscope Studies., Return to: Contents.