Transcript Document
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/~rhodes/CMCC/index.html
http://ruppweb.dyndns.org/Xray/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.berkeley.edu/~jamesh/movies/
Cool movies demonstrating key points about diffraction, resolution, data quality, and refinement.
Practical Applications of X-ray Crystallography
Crystal -> Diffraction pattern -> Electron density -> 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?
The Crystal Lattice
The Seven Crystal Systems
The 14 Bravais Lattices
Crystals and the asymmetric unit:
Crystals and the asymmetric unit:
Rotational Symmetry Operators:
Rotational Symmetry Operators:
Translational Symmetry Operators:
Translational Symmetry Operators:
How are molecules packed within the lattice?
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
Techniques for super-saturation and growth
Batch crystallization
Liquid-liquid diffusion
Dialysis
Vapor-diffusion: Hanging drop or sitting drop
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Once you have crystals..
Are they what you want?
Do they diffract?
Can they be cryo-cooled?
Data processing
Determine your space group. Index and integrate.
h,k,l, I
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
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?
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?
What does resolution mean in practice?
6.0 Å
4.5 Å
3.0 Å
1.6 Å
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
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