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Browsing by Subject "Coot"

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    Purification, crystallization and structure determination of an azurin variant.
    (2012-08) Ladd, Melanie Ann
    X-ray diffraction is a method that allows the three-dimensional structure of a molecule to be determined. To use this technique to study a protein model, high-quality crystals were grown. A biosynthetic approach was taken to model the mammalian protein peptidylglycine α-hydroxylating monooxygenase (PHM), which is a copper-binding protein that hydroxylates the α-carbon of a glycine residue in the production of peptide hormones. In order to understand the mechanism of this reaction, a model of the two copper sites involved in hydroxylation was created using the bacterial protein azurin as a scaffold (Az-PHM). To compare the structural similarity of the model to the native PHM system, Az-PHM crystals were grown for x-ray diffraction using various buffers, salts, polyethylene glycol (PEG) and excess copper. Dozens of the resulting crystals were diffracted, which had lower resolutions (~2.5 Å) and higher mosaicities (0.8 - 1.2° on average). Crystal dehydration and cryoprotection techniques were applied and consistently yielded higher resolution and lower mosaicity crystals. The crystal with the highest resolution and low mosaicity was grown in Tris buffer, lithium nitrate, PEG-2000 and copper chloride. Diffraction images for this crystal were collected on a Rigaku RAPID II X-ray Diffractometer using a copper radiation source with capillary optics and an R-AXIS image plate detector. Data were indexed to yield a P212121 space group, which was then followed by integration, scaling and averaging using CrystalClear 2.1 software. Phases were determined using the Molecular Replacement method in the software CCP4. Finally, structural refinement of the model and electron density map in Coot yielded a 1.3 Å structure with an Rfactor of 17.57% and an Rfree of 20.70%.