Structural Studies of a Urea Channel with Electron Microscopy

The Urea/Amide channel from Bacillus cereus (UACBc) was expressed in Escherichia
coli with a C-terminal hexa-histidine tag. The protein was purified in detergent as
confirmed by N-terminal sequencing. The purified protein in detergent was analysed
with single particle analysis processing and forms a particle consisting of a pair of
stacked discs with diameters of 120 Å with each disc representing an oligomer of
UACBc.
Two-dimensional (2D) crystallisation produced highly aggregated crystals that
became suitable for high resolution imaging upon sonication to disperse them.
Using the 2D crystals for electron cryomicroscopy yielded images that upon
crystallographic processing and analysis suggested that the crystals had p6 symmetry
with an additional single p622 crystal indicating a possible double-layered crystal
form.
The images with p6 symmetry were merged to produce a 9 Å projection map showing
the protein forming a hexameric ring with 7 density features in each putative
monomer possibly representing the predicted 7 transmembrane helices of UACBc.
AFM and production of a negative stain three dimensional (3D) density map were
used to determine the thickness of the crystals and based on a mono-layered crystal
form, bioinformatic analysis and biochemical experiments to verify the oligomeric
state and topology, a model with the putative locations of the 7 predicted
transmembrane helices and their orientations with respect to each other has been
produced.