The X-ray crystallography Core provides a state-of-the-art facility for the generation of crystals and structure determination of macromolecules including proteins, DNA, RNA, and complexes between macromolecules and their ligands.  To complement structural data, the core provides state-of-the-art instrumentation to measure the affinities and kinetics of macromolecules with ligands and/or other macromolecules.  Finally, the core has assembled a 1250 member, fragment library to screen for potential lead compounds through differential scanning fluorimetry, SPR, ITC and diffraction methods.
Sample Analysis and Preparation
Consultation and in silico analysis
  • Protein expression and purification (bacteria, insect or mammalian cell expression/ affinity or tag-less purification)
  • Domain structure, including disorder and secondary structure prediction
Physical Analysis
  • Native and SDS PAGE
  • Limited Proteolysis and characterization by SDS-PAGE
  • Size Exclusion Chromatography (analytical and preparative grade)
  • Sedimentation Equilibrium analysis by Analytical Ultracentrifugation (AUC)
  • Sedimentation Velocity analysis by AUC
  • Surface Plasmon Resonance (SPR)
  • Circular Dichroism (CD) Spectroscopy
  • Isothermal Titration Calorimetry (ITC)
  • Kinetic Exclusion Assay (KinExA)
  • Differential Scanning Fluorimetry (DSF)
  1. Set up crystallization trials at 4 ºC and/or 20 ºC.
    1. Initial trials (e.g., appropriate concentrations)
    2. Full scale trials (4 different 96 well factorials at 3 protein concentrations and 2 temperatures)
  2. Optimization – additive screens, factorial overlays, macro and micro seeding
  3. Automated imaging available for4 ºC and 20 ºC )
Diffraction quality
  1. Test diffraction quality (loop and capillary mounting available)
  2. Test/screen cryo-conditions
Data collection
  1. Collect, reduce and merge data. Generate table of statistics
  2. MAD/SAD phasing – help design, collect, reduce and merge MAD data
  3. Generate table of statistics including anomalous dispersion differences
Structure Determination
  1. Solve structure by Molecular Replacement
  2. Solve structure by MAD/SAD phasing
  3. Refine structure
  4. Produce relevant statistics (e.g., R and Rfree, RMS deviations)
  5. Structural analysis (superpositions, electrostatics, etc)
  6. Deposit Structure at PDB