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Nobel Laureates of X Ray Crystallography. Max von Laue - 1914 Nobel Prize in Physics Bragg(s) - 1915 Nobel Prize in Physics. Dorothy Hodgkin – 1964 Nobel Prize in Chemistry Hauptman and Karle - 1985 Nobel Prize in Chemistry Roderick MacKinnon and Peter Agre – 2003 Nobel Prize in Chemistry. - PowerPoint PPT Presentation
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Nobel Laureates of X Ray Crystallography
• Max von Laue - 1914 Nobel Prize in Physics
• Bragg(s) - 1915 Nobel Prize in Physics.
• Dorothy Hodgkin – 1964 Nobel Prize in Chemistry
• Hauptman and Karle - 1985 Nobel Prize in Chemistry
• Roderick MacKinnon and Peter Agre – 2003 Nobel Prize in Chemistry
Cα
Cα
ψ
φ
Levinthal's paradox
• How many backbone conformations of a 300 residue protein are possible?
• Only taking into account Phi and Psi angles, approximately 10³°° conformations
• How is the right conformation found in our lifetime?
• Answer: Only some angle combinations are energetically favorable, hence only a limited amount of structural conformations are possible
Steps To Solving The Structure Of A Protein
• When X-rays strike a macromolecular crystal, the atoms in the molecules produce scattered X-ray waves which combine to give a complex diffraction pattern consisting of waves of different amplitudes
• What is measured experimentally are the amplitudes and positions of the scattered X-ray waves from the crystal
• X-ray crystallography provides the positions of all non-hydrogen atoms
• The origin of each wave must be determined so that they sum to give an image instead of a “sea of noise”
• Phase values must be assigned to all of the recorded data; sometimes done computationally, but is usually done experimentally by labeling the protein with one or more heavy atoms whose position in the crystal can be determined independently
Electron Density Calculation
• Diffraction amplitudes = FT{Electron density}• Take the inverse FT of the diffraction pattern to
regenerate the electron density• Shooting a crystal with X-rays and obtaining a
diffraction pattern is the same as taking the Fourier transform of a compound. Hence, taking the Fourier transform again gives us the original structure.
Our Protein
After shootingOur Protein with X-raysAnd getting the FT
Taking the FT of the FTRESTORES the originalData (mostly)
A very simple example of Fourier and Inverse Fourier transforms
• The scattered x-rays have amplitudes given by Fourier coefficients of electron density
• Possible to measure amplitudes• If we could also measure phases, we could
compute electron density by inverse Fourier transform
• We then fit a model to the density• Phases are extremely difficult to measure
Quick Recap
• Crystallize Protein (if humanly possible)
• Measuring diffraction amplitudes
• Using a computer to calculate electron density
• Building a model consistent w/ density
Quality Of a Structure
• R-factors represent the percentage disagreement between the observed diffraction pattern and that calculated from the final model
• R-factors of around 20% or less are considered well determined structures that are expected to contain relatively few errors
• Express the resolution of a structure in terms of a distance
Molecular Replacement
• Use an analogous structure (similar amino acid sequence) and apply to the structure you are trying to determine
• “Replacement” actually means 'relocation, repositioning‘ atoms.
(Multiple) Isomorphous Replacement