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bme 140 galburt - IMAGING MANIPULATING BIOMOLECULES Eric...

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IMAGING & MANIPULATING BIOMOLECULES Eric Galburt [email protected] BME 140 10 November 2010
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Structural Analyses Manipulation and Measurement METHODS What does the molecule look like? How do the parts fit together? How to the parts move relative to one another? What are the material properties? How do biomolecules respond to forces? The advantages of single molecule measurements.
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Structural Analysis X-ray crystallography electron microscopy (EM) atomic force microscopy (AFM) nuclear magnetic resonance spectroscopy (NMR)
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ppt + protein X-RAY CRYSTALLOGRAPHY ppt h2o obtaining protein crystals via the hanging drop method screen hundreds to thousands of buffer conditions, temperatures, protein concentrations, etc... Rigaku’s crystalmation
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X-RAY CRYSTALLOGRAPHY Galburt and Stoddard, Physics Today 2001
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X-RAY CRYSTALLOGRAPHY X-ray beam crystal detector diffracted beam
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X-RAY CRYSTALLOGRAPHY
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X-RAY CRYSTALLOGRAPHY X-ray beam detector diffracted beam diffraction grating λ = wavelength d = spacing θ = angle m = order http://www.mwit.ac.th/~Physicslab/
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X-RAY CRYSTALLOGRAPHY The Fourier transform relates the intensities of the diffraction image to the actual electron density. Need both the intensities from the diffraction pattern AND the phases of each reflection.
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X-RAY CRYSTALLOGRAPHY http://www.xtal.iqfr.csic.es/Cristalografia/ interpretation of electron density leads to the structure
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X-RAY CRYSTALLOGRAPHY examples of typical resolutions for a tyrosine residue http://www.pdb.org
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X-RAY CRYSTALLOGRAPHY T4 polynucleotide kinase what roles to individual residues play in binding and catalysis? how is the primary sequence arranged in space? Galburt and Stoddard, Structure 2002
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X-RAY CRYSTALLOGRAPHY time resolved crystallography uses multiple wavelengths from synchrotron radiation (Laue diffraction) and short exposure times (ns) after laser initiated reactions to collect dynamic information LAUE diffraction, Argonne National Labs CO-myoglobin, Schotte and Anfinrud, Science (2003)
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ELECTRON MICROSCOPY wikipedia
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ELECTRON MICROSCOPY JEOL 3100, Nogales lab website
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CRYO-ELECTRON MICROSCOPY uses electrons instead of photons uses magnetic fields as lenses based on the de Broglie wavelength of the electrons (on the order of picometers, dependent on the velocity of the electron/voltage of the electron gun) GroEL protein chaperone at 50,000X from wikipedia MUCH higher resolution that light microscopy (5-50 Å) images are PROJECTIONS!
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CRYO-ELECTRON MICROSCOPY take many images of particle projections at random orientations
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