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Build model and refine

Model Building and Refinement for CHEM 645. Purified Protein. Solve Phase. Build model and refine. | F hkl | = ( I hkl ) 1/2. phase angle a hkl. Decompose F hkl. real space x = Å. reciprocal space h = Å -1. FT. Multi-wavelength Anomalous Dispersion (MAD phasing).

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Build model and refine

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  1. Model Building and Refinementfor CHEM 645 Purified Protein Solve Phase Build model and refine

  2. |Fhkl| = (Ihkl)1/2 phase angle ahkl Decompose Fhkl real space x = Å reciprocal space h = Å-1 FT

  3. Multi-wavelength Anomalous Dispersion (MAD phasing) Se, Hg, Yb, Ho, etc… XAFS – x-ray absorption fine structure, measure by fluorescence intensity off of protein crystal as a function of x-ray energy

  4. MAD phasing - continued Example: Isocitrate Dehydrogenase (IDH) click here for pdf of structure paper • Dimer of 46,500 Da subunits • - 9 methionines/subunit • Selenomethionine expression (Sel-met) experimentally measured MAD-phases measured amplitudes

  5. MAD-phased map at 3.1 Å of IDH • Initial map of IDH showed clear • molecular boundaries • Allows phase improve technique • called Solvent Flattening • make mask around IDH mol. • assign an average value to • rho in solvent channels • “correct” the Fhkl using the • corrected function of rho. this would have been nicer

  6. One dimer of IDH – MAD phased map with bones built in Interpret map – from bones, find a-helices, b-sheets, trace chain NC, add side chains, and refine. This is your initial model!

  7. Obtain |Fcalc| and acalc from Initial Model Initial model build in theoretical reciprocal space X-ray Refinement |Fcalc| and acalc ideal geometry term x-ray term  = wFwhkl (|Fo| - |Fc|)2hkl + wE Etotal hkl calculated observed

  8. Observing Comparison Between |Fcalc| and |Fobs| using Electron Density Difference Maps Experimentally observed calculated calculated from current model Shorthand: Fo - Fc difference maps + where model should be added - where model should be removed 2Fo - Fc Fo - Fc

  9. Active Site and Complete Model of IDH 2Fo – Fc map

  10. How do you know when a structure is done? R-factor = |Fobs| - |Fcalc| |Fobs| R-free - set aside 10% of reflections not used in refinement or map generation, use only to evaluate Other criteria: Minimal Deviations from Ideal Geometry RMSD < 0.01 Å bond lengths < 2 º bond angles Ramachandran plots of phi and psi

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