Model Building and Refinement for CHEM 645

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Presentation transcript:

Model Building and Refinement for CHEM 645 Purified Protein Solve Phase Build model and refine

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

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

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

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

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!

 = wF  whkl (|Fo| - |Fc|)2hkl + wE Etotal 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

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

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

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