Structure Determinaton

Structure Determinaton

1. X Ray

   • Crystals
     • Production
       • Droplet over precipitant
       • Trial and error
         • Roboticaly
       • Empical search
       • Make recombinantly
       • Nucleate
     • Selected against
       • Hard
   • Diffraction
     • Poor crystals
       • 50% solvent
       • Ineffective
     • Braggs law
       • Dots formed
       • Contain data
       • Close dots = far away
       • Large dots far away = high crystal
       • Rotate crystal to get all points
     • Intensity is key
   • Analyasis
     • Structure factor
       • Constant value
       • Known
     • Phase component by guess
     • Calculates density map
   • Model
     • Best guess of map
     • All atoms built into mesh
     • 1.5+ A map
     • Now automated
     • Very robust method
   • No H atoms

2. NMR

   • Very physicsy
   • Method
     • RF in solution
     • Spin sensitive absorbance
     • Look for resonance
     • Determines chemical envioment
     • See H atoms
       • C13/N15 (rare)
   • Issues
     • Need recombinant tagged protien
   • Data
     • Many different peaks
     • Label to differentiate by atom
     • Nuclear overhouser shows distance
     • Create rules
     • Model to rule satisfacton
     • Solutions
       • Many equal likely posibilites
       • Outer conformations undetermined
       • Dynamic images
   • Small things
   • Dynamic imaging

3. EM

   • TEM
     • Direct imaging
     • Focused image produced
     • Low contrast
   • Mechanism
     • Freeze
     • Take many images
     • Reconstruct full image
   • Big things
   • Issues
     • Low resolution
     • Low contrast
     • Thousands of identical images needed
   • Small changes = very wrong
   • Progress
     • Now resoluion about Xray
     • High speed CCD's
     • Computing horsepower

4. Folds

   • 1000's
     • Many less than sequences
     • Highly conserved
   • Structure > Sequence conservation
   • Sequence changes
   • AB topology is important
   • Helical
     • Soluble
       • Hb / Mb
     • Membrane
       • Selective
     • GPCR's
   • Beta
     • Beta barrels

5. Non globular

   • Fibourous protiens
     • Hair
     • Skin
   • Coiled coil
     • Amphipathic helix
     • Hair
   • Collagen
     • 1/4 mammals
     • Hydroxyproline (vitC)
     • High sequence repeat
       • Wrap round each other
       • Strong