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Methods for structural elucidation

Methods for structural elucidation. Protein crystallization. pH Ionic strength Temperature Concentration of precipitant Concentration of macromolecule Purity of macromolecule Additives, effectors, and ligands Organism source of macromolecule Substrate, coenzymes, inhibitors

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Methods for structural elucidation

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  1. Methods for structural elucidation

  2. Protein crystallization

  3. pH Ionic strength Temperature Concentration of precipitant Concentration of macromolecule Purity of macromolecule Additives, effectors, and ligands Organism source of macromolecule Substrate, coenzymes, inhibitors Reducing or oxidizing environment Metal ions Rate of equilibrium Surfactants or detergents Gravity Vibration and Sounds Volume of crystallization Sample Presence of amorphous material Surface of crystallization vessels Proteolysis Contamination by microbes Pressure Electric and magnetic fields Handling by investigator Factors Affecting Protein Crystal Growth

  4. Bragg’s Law: nl=2dsin

  5. e- Concept 1: Some nuclei have non-zero spin quantum numbers. Nuclei with odd mass numbers have half-integer spin quantum numbers. i.e. 13C, 1H, 31P are spin I = 1/2 17O is spin I = 5/2 Nuclei with an even mass number and an even charge number have spin quantum numbers of zero. ie. 12C Nuclei with an even mass number and an odd charge number have integer spin quantum numbers. i.e. 2H is spin I = 1 Electrons also have a spin quantum number of 1/2

  6. Concept 2: Current passed through a coil induces a magnetic field. e- e- Concept 3: A changing magnetic field in a coil induces a current. e- e-

  7. QM picture Concept 4: Placing nuclei with spin I = 1/2 into a magnetic field leads to a net magnetization aligned along the magnetic field axis. Mz Classical picture

  8. Large external magnet Net magnetization aligned along Z-axis of the magnetic field Bo

  9. The B1 field is produced by a small coil in the NMR probe which is placed in the bore of the large external magnet. B 1 Bo Net magnetization aligned along x-axis of the magnetic field after application of B1 field.

  10. NMR magnet. B 1 Bo NMR probe e- e-

  11. Concept 5: When the B1 field is turned on, the net magnetization rotates down into the XY plane z Bo x y

  12. z T 1 Bo x y Concept 6: When the B1 field is turned off, the net magnetization relaxes back to the Z axis with the time constant T1 T1 is the “longitudinal” relaxation time constant which results from “spin-lattice” relaxation

  13. MAS NMR Static 13CH2 13COOH 512 scans 10 kHz MAS 4 scans Chemical Shift MAS improves resolution and sensitivity

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