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.
Sample Analysis:
Consultation andin silicoanalysis
  • Protein expression and purification
  • Domain structure, including disorder and secondary structure prediction
Physical Analysis
  • Native PAGE using GE HealthSciences PhastGel
  • Limited Proteolysis and characterization by SDS-PAGE
  • Size Exclusion Chromatography
  • Sedimentation Equilibrium analysis by AUC
  • Sedimentation Velocity analysis by AUC.
  1. Set up crystallization trials at 4 ºC and/or 20 ºC.
    a.Initial trials (e.g., appropriate concentrations)
    b.Full scale trials (4 different 96 well factorialsat 3 protein concentrations and 2 temperatures)
  2. Optimization – additive screens and factorial overlays
  3. Automated Screening at 2 temperatures (scan daily forfirst week, weekly thereafter, finish experiment at 3 months)
Diffraction quality:
  1. Test diffraction using capillary mounted crystal
  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