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The method of Multiple wavelength Anomalous Diffraction using Synchrotron Radiation at optimal X-ray energies:
Application to Protein Crystallography.

• Contents
• Introduction.
  • Protein Crystallography.
    • Protein structure.
    • Protein crystal structure determination.
  • Multiple wavelength methods.
    • Experimental requirements of the MAD method.
  • Outline of the thesis.
• Theory and methods of data analysis.
  • X-ray diffraction.
    • Classical X-ray scattering.
    • Anomalous scattering.
      • Classical description of dispersion.
      • Absorption and the complex refractive index.
    • Diffraction from a crystal.
    • Effects of anomalous scattering on the structure factor equation.
    • Phase problem.
  • Determination of the heavy atom positions.
    • Patterson methods.
    • Difference Fourier techniques for locating heavy atoms.
  • Direct methods applied to the solution of the heavy atom structure.
    • Underlying theory.
    • MULTAN software package.
    • The convergence method.
    • The magic integer method.
    • Figures of merit.
    • Application to the solution of the heavy atom structure.
  • Multiple wavelength Anomalous Diffraction.
    • Classical approach.
      • Treatment of errors in the classical approach to MAD phasing.
    • Algebraic approach.
  • Analysis of diffraction image data collected using the rotation method.
    • The rotation method.
    • Orientation refinement and reflection integration.
      • Orientational refinement.
      • Integrated intensities by profile fitting.
      • Lorentz-Polarisation corrections.
    • Image scaling and merging.
    • Scaling of data from different wavelengths.
    • Calculation of phases.
    • Phase improvement and density modification.
    • Quality of electron density maps.
    • Model refinement.
  • Evaluation of anomalous scattering factors, and .
• Experimental Apparatus.
  • DORIS III storage ring.
  • X31 beam line.
    • Monochromator.
    • Photo-emission device.
    • Fluorescence detection.
    • Absolute energy calibration and monitoring.
      • Introduction.
      • Theory of the calibrator.
      • Construction of the calibrator.
      • Operation of the calibrator.
      • Using the calibrator to monitor X-ray energy stability.
    • X31 beam line characteristics.
      • Intrinsic resolution of silicon (111) and (311) reflections.
      • Measurements using the Si(111) monochromator.
      • Measurements using the Si(311) monochromator.
      • Contributions to X-ray energy instability.
  • X11 beam line.
    • X11 beam line characteristics.
  • Image plate detector.
    • The image plate phosphor.
    • Image readout and correction.
    • Sources of error in the detector.
• MAD experiments.
  • Introduction.
  • Third transition series edges.
  • MAD experiments on an Iridium derivative of Lysozyme.
    • Crystallisation and derivative preparation.
    • Derivative screening.
    • X-ray energy selection via direct fluorescence measurements.
    • Data collection.
  • Data analysis.
  • Solution of the heavy atom structure with MULTAN.
  • Lysozyme structure refinement.
    • Native structure.
    • Derivative structure.
  • MAD phasing of X31 data.
  • MAD phasing of X11 data.
  • Agreement between experimentally determined and refined anomalous scattering factors.
• Discussion.
  • X-ray energy calibration apparatus.
    • Absolute energy calibration of fluorescence spectra.
  • Experimental procedures in MAD.
  • Anisotropy of anomalous scattering.
  • Phasing procedures.
  • Conclusions.
• References
• Index
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