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Title: Structural studies of a urea channel with electron microscopy
Author: Chan, Nathan
ISNI:       0000 0004 2744 033X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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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.
Supervisor: Bullough, Per Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available