Structure determination

• Primary structure can be determined using mass spec. or Edman Degradation
• Secondary structure can be determined with circular dichroism, IR, and NMR
• Tertiary and quartnary structure can be determined by x-ray crystallography, cyro-electron microscopy, and NMR
• Small angle x-ray scattering: models a protein's shape based on scattering but with very low resolution
• X-ray crystallography requires knowledge of the amino acid's sequence to create a protein image.

  x-ray -> crystal lattice -> diffraction pattern -> e- density calculation

  -> image creation
• Protein crystallization must undergo micro dialysis in order to slowly remove water using salt and to not denature the protein.

  Proteins undergoing must also be highly structured, so excludes disordered regions
• At 2.5-3 Armstrong, you can see phi psi constraints
• Due to the resolution of x-ray crystallography (2.5-3 Armstrong) you cannot tell the difference between C, N, or O.

  Asn, Asp, and Leu all would look the same
• NMR is difficult for molecules bigger than 40 kDa but good that they can be in solution
• For cyro-electron microscopy, the sample must be cooled to ~ -196°C very rapidly to remain hydrated.

  Good for big molecules with a resolution of >3 armstrong
• SAXS pros

  • done in solution (no crystal)
  • small sample needed
  • relatively fast

  X-ray crystallography

  • well developed
  • high resolution
  • wide range of MW's

  NMR

  • high resolution
  • in solution
  • dynamic info

  Cyro-Electron microscopy

  • easy sample prep
  • native state
  • small sample size
• SAXS cons:

  • low resolution (~30 armstrong)
  • only good at analyzing large complexes

  X-ray crystallography

  • difficult crystallization
  • static structure
  • damaging x-rays

  NMR

  • high sample purity needed
  • protein must be small (<40 kDa)

  Cyro-electron microscopy

  • low relative resolution
  • only best for high MW proteins