Introduction to Macromolecular Xray Crystallography

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Introduction to Macromolecular X-ray
Crystallography Biochem 300 Borden Lacy
Print and online resources Introduction to
Macromolecular X-ray Crystallography, by
Alexander McPherson Crystallography Made Crystal
Clear, by Gale Rhodes http//www.usm.maine.edu/rh
odes/CMCC/index.html http//ruppweb.dyndns.org/Xr
ay/101index.html Online tutorial with interactive
applets and quizzes. http//www.ysbl.york.ac.uk/
cowtan/fourier/fourier.html Nice pictures
demonstrating Fourier transforms http//ucxray.be
rkeley.edu/jamesh/movies/ Cool movies
demonstrating key points about diffraction,
resolution, data quality, and refinement.
http//www-structmed.cimr.cam.ac.uk/course.html No
tes from a macromolecular crystallography course
taught in Cambridge
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Practical Applications of X-ray Crystallography
Crystal -gt Diffraction pattern -gt Electron
density -gt Model
Spacegroups, obtaining crystals Data collection
and related statistics Evaluating structures and
reading structure papers What can you see and
learn with this method? What else can you do?
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The Crystal Lattice
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The Crystal Lattice
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The Seven Crystal Systems
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The 14 Bravais Lattices
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Crystals and the asymmetric unit
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Rotational Symmetry Operators
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Rotational Symmetry Operators
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Translational Symmetry Operators
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Translational Symmetry Operators
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How are molecules packed within the lattice?
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How to obtain crystals
Work with sample that you expect will be
structured.
Obtain pure, homogeneous sample at high
concentration
Bring your sample solution to supersaturation to
allow for spontaneous nucleation by varying the
pH, temperature, and/or concentration of salt,
precipitant, and/or organic solvent.
Allow crystals to grow.
mosaicity
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Techniques for super-saturation and growth
Batch crystallization Liquid-liquid
diffusion Dialysis Vapor-diffusion Hanging drop
or sitting drop
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Once you have crystals.. Are they what you
want? Do they diffract? Can they be cryo-cooled?
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Data processing
Determine your space group. Index and integrate.
Merge partial reflections, determine scale
factors that minimize differences between
identical reflections collected on different
frames, and average the intensities of
symmetrically identical reflections. h,k,l, I, s
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Assessing data quality
Completeness, redundancy, signal-to-noise (I/s),
how well do different frames of data agree after
scaling (Rmerge)? How do these statistics look
for the high-resolution data?
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How good are your maps?
Molecule-solvent separation Continuous density,
secondary structure Features consistent with the
resolution of the data ( ex. side chains, ordered
water) Can you omit pieces of the model and still
see them due to your phases?
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What does resolution mean in practice?
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Criteria for judging a structure
R-factor and Rfree Deviation from ideal bond
lengths and angles Distribution of y and f
peptide bond torsion angles
Ramachandran Plot
Consistency with previously determined
structures Chemical and biological sense
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What else can you do? Membrane proteins Complexes
Drug design Structural genomics Laue diffraction
and time resolved crystallography Neutron
diffraction Metallochemistry with XAFS Small
angle x-ray scattering Fiber diffraction