To do the EM shown
here, preparations of purified VacA molecules
were adsorbed to a suspension of finely ground
mica flakes, followed by freeze-drying and
platinum replication according to established
procedures (refs. 1,2). (See also ref.3 for
earlier images of VacA prepared identically.)
Briefly, this preparation involved adding
two drops of a suspension of finely ground
mica flakes to 0.5 ml of a solution containing
10-30 ug purified VacA, and allowing the
toxin to adsorb to the mica for 30 sec.
The mica flakes then were pelleted by
gentle centrifugation, washed twice with
a solution of 30 mM Hepes (pH 7.2) containing
70 mM KCl and 5 mM MgCl2, and layered
onto a thin slice of aldehyde-fixed lung
for support during freezing. Freezing
was accomplished by dropping the samples
onto a liquid helium-cooled copper block
within a homemade quick-freezing device.
Quick-frozen mica flakes were freeze-fractured
in a Balzer's freeze etch machine and
then etched for 4 min at -100oC. Exposed
molecules adsorbed to mica were rotary-replicated
with ~2 nm of platinum applied from an
angle of 11 degrees above the horizontal,
and finally "backed" with a
10 nm support film of pure carbon. The
replicas were separated from mica by immersion
overnight in concentrated hydrofluoric
acid, washed in water, and finally picked
up on 75-mesh formvar-coated microscope
grids. They were viewed in a JEOL transmission
electron microscope operated at 100 kV,
and photographed in stereo using +10 degrees
of tilt with a eucentric side-entry goniometer
stage. Finally, representative molecules
were chosen by video microscopy from micrographs
initially taken at 68,000x, video images
were transfered to a computer, and therein
they were enlarged to 300,000x for final
generation of the photo-montages presented
here, using Adobe Photoshop.
- Heuser, J.E. 1983. Procedure for freeze-drying
molecules adsorbed to mica flakes.
J. Mol. Biol. 169: 155-195.
- Heuser, J.E. 1989. Protocol for 3-D
visualization of molecules on mica
via the quick-freeze, deep-etch technique.
J. Elect. Micro. Tech. 13: 244-263.
- Lupetti, P., J.E. Heuser, R. Manetti,
P. Massari, S. Lanzavecchia, P.L. Bellon,
R. Dallai, R. Rappuoli and J.L. Telford.
1996. Oligomeric and subunit structure
of the helicobacter pylori vacuolating
cytotoxin. J. Cell Biol. 133:801-807