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SINGLE PARTICLE IMAGE RECONSTRUCTION: 2D TO 3D IN CRYO-EM
Restricted (Penn State Only)
Feinberg, Mandi Alysson
Master of Science
Date of Defense:
March 23, 2017
Susan Hafenstein, Thesis Advisor
Acetyl-CoA decarbonylase/ synthase
Cryo-electron microscopy (cryo-EM) has begun to emerge as the front-runner for obtaining atomic resolution structures of protein complexes. The work presented here shows the multiplicity of cryo-EM with a reconstruction of a small, asymmetric protein complex and a larger, icosahedrally symmetric virus. Acetyl-CoA decarbonylase/synthase (CDH) is an anaerobic, bifunctional enzyme complex in M. acetivorans. Its enzymatic activities include CO dehydrogenase and acetyl-CoA synthase. CDH is key in the conversion of acetate to methane, which accounts for two-thirds of Earth’s biologically produced methane. This 258 kDa complex is comprised of five subunits, α,β,γ,δ, and ε, which together, make up three functional components, Cdhβ, Cdhαε, and Cdhγδ . Structures of components of CDH-like proteins from distant relatives to M. acetivorans have been solved though no further structures for the entire CDH complex or components have been determined. A preliminary 27 Å resolution CDH structure was reconstructed from a small data set imaged on an Ultrascan 4000 charge-coupled-device (CCD) camera in a JEOL 2100 LaB6 cryo-EM microscope. The preliminary structure shows features indicative of CDH function, including a channel where CO is transferred between subunits, thus confirming the ability to obtain a structure of CDH through cryo-EM. In order to improve upon the resolution of the preliminary structure, a data set taken on a DED microscope is currently being processed. The end result will be a high resolution structure of CDH in which structural features of each domain can be recognized and determined. Obtaining the structure of CDH is revolutionary to the field of study and will allow for studies of CDH that have been inconceivable without a complete structure to be possible. Canine parvovirus (CPV) is a member of the Parvoviridae, a family composed of non-enveloped viruses around 25 nm in diameter and containing a single-stranded DNA genome. Though CPV is a systemic disease, it typically manifests as enteritis in canines between the ages of six weeks and six months. The CPV capsid displays a T=1 icosahedral symmetry and is assembled from 60 copies of a major capsid protein, consisting of 90% viral protein 2 (VP2) and 10% of a minor capsid protein, VP1. In wild type CPV, 10% of the VP2 in a virus capsid gets cleaved between residues Asp269 and Cer270. These residues sit on the internal, three-fold axis of the capsid. The proteolytic activity that results in a cleavage at this site is unclear. By mutating residue 270 from a cysteine to a serine, the cleavage event does not occur and results in a 100-fold decrease in virus production during infection. Therefore, in order to perform additional tests, a virus-like particle (VLP) of the mutant and wild type were made and reconstructed. A 5.41 Å preliminary reconstruction of CPV-VLP mutant and a 7.73 Å preliminary reconstruction of CPV-VLP wild type resulted from data sets imaged on a FEI Falcon 2 DED installed on a FEG Polara G2 microscope. When a CPV wild type crystal structure was fit into the mutant preliminary reconstruction, there was no noticeable change at the three-fold of the virus where residue 270 resides. The data are still being processed to cut the particles down to the best of the best to obtain a high resolution structure for both VLPs. Obtaining a high resolution reconstruction of both mutant and wild type VLPs will allow for a direct comparison of the two structures. A localized reconstruction of the inner three-fold of these two structures may allow for a better understanding for the asymmetric cleavage event as well as why this even will not occur with the Cys270Ser mutation.
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