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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.

  1. Heuser, J.E. 1983. Procedure for freeze-drying molecules adsorbed to mica flakes. J. Mol. Biol. 169: 155-195.
  2. 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.
  3. 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
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