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Imaging and 3D reconstruction of membrane protein complexes by cryo-electron microscopy and single particle analysis

Gregorini, Marco. Imaging and 3D reconstruction of membrane protein complexes by cryo-electron microscopy and single particle analysis. 2006, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7966

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Abstract

Cryo-electron microscopy (cryo-EM) in combination with single particle image processing and volume reconstruction is a powerful technology to obtain medium-resolution structures of large protein complexes, which are extremely difficult to crystallize and not amenable to NMR studies due to size limitation. Depending on the stability and stiffness as well as on the symmetry of the complex, three-dimensional reconstructions at a resolution of 10-30 ˚ can be achieved. In this range of resolution, we may not be able to answer A chemical questions at the level of atomic interactions, but we can gain detailed insight into the macromolecular architecture of large multi-subunit complexes and their mechanisms of action. In this thesis, several prevalently large membrane protein complexes of great physiological importance were examined by various electron microscopy techniques and single particle image analysis. The core part of my work consists in the imaging of a mammalian V-ATPase, frozen-hydrated in amorphous ice and of the completion of the first volume reconstruction of this type of enzyme, derived from cryo-EM images. This ubiquitous rotary motor is essential in every eukaryotic cell and is of high medical importance due to its implication in various diseases such as osteoporosis, skeletal cancer and kidney disorders. My contribution to the second and third paper concerns the volume reconstruction of two bacterial outer membrane pore complexes from cryo-EM images recorded by my colleague Mohamed Chami. PulD from Klebsiella oxytoca constitutes a massive translocating pore capable of transporting a fully folded cell surface protein PulA through the membrane. It is part of the Type II secretion system, which is common for Gram-negative bacteria. The second volume regards ClyA, a pore-forming heamolytic toxin of virulent Escherichia coli and Salmonella enterica strains that kill target cells by inserting pores into their membranes. To the last two papers, I contributed with cryo-negative stain imaging of the cell division protein DivIVA from Bacillus subtilis and with image processing of the micrographs displaying the siderophore receptor FrpB from Neisseria meningitidis.
Advisors:Engel, Andreas
Committee Members:Boisset, Nicolas
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Engel)
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7966
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:89
Language:English
Identification Number:
Last Modified:23 Feb 2018 11:43
Deposited On:13 Feb 2009 16:09

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