We use a CM12 transmission Electron Microscope from
Philips.
It's equipped with Gatan cryo
add-ons to work with frozen samples.
We study structure of
E. coli RNA polymerase using Electron Crystallography.
In the presence of positively charged lipids, core RNA polymerase
molecules form tubular structures. These structures are made of
lipid bilayer tubes ~ 1000 A in diameter with RNA polymerase molecules
sitting on the outer surface of these tubes in helical symmetry.
During negative staining the hollow tubes flatten and form two layers of
two-dimensional crystals.
The 3D structure of core RNA polymerase
was solved by computer processing of the images of such
2D crystals tilted 0 - 60 degrees to the incident electron beam.
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The tubes preserved in negative stain (top) reveals their well-ordered structure at about 35,000 magnification. Both sides of the flatten tube are visible.
At lower magnification (~ 2,500) they appear as long
thin structures growing from the surface of lipid vesicles.
The helical crystals of core RNA polymerase preserved in negative stain and shadowed with Platinum are in the middle. This kind of shadowing provides a view of only the top side of a crystal. You clearly see that crystals are right-hand helices. Helical crystals preserved in amorphous ice, exhibit helical diffraction. |
The helical crystals of core RNA polymerase here hold great promise for the determination of a more informative RNAP structure from specimens preserved in the frozen-hydrated state and studied by the method of cryo-EM (DeRosier and Moore, 1970;
Dubochet et al., 1988). Such a structure
would
Additional information on core RNA polymerase structure, crystallization
conditions and electron crystallography can be found in our recent paper
(Polyakov et al., Cell 1995, 83, pp 365-373).
