Solid State NMR

Structure and dynamics of bio-solids

 

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Solid State NMR Details

User Guide

 

Solid State NMR spectroscopy measurements can be performed in virtually any kind of biological solid. 13C-15N labelled samples are required, since the vast majority of the experiments are based on direct heteronuclear detection. Recently, 2H labelled proteins back exchanged in water are becoming popular, since they allow for proton detection. Local order in the solid as well as its hydration state are known to dramatically affect spectral quality; micro- to nanocrystalline proteins are therefore the target of these studies.

Magic angle spinning is used to average the anisotropic interactions, so the sample is packed in zirconia rotors from 1.3 to 4 mm. The amount of sample ranges from about 2 mg in the 1.3 rotor to 50 mg in the 4 mm rotor. Despite the much smaller amount of material, the signal to noise ratio in the 1.3 rotor can be almost as good as in larger rotors, because, among other factors, of the higher efficiency of the smaller coils. Since the RF heating in SSNMR is more severe than in solution, low salt samples are required.

The acquisition of spectra takes from a few minutes (1D spectra) to a few days (3D spectra), depending on the amount of sample and its quality.

Once the sample is placed in the probehead and the desired temperature and spinning rates have been reached, the pulses are calibrated and polarization transfer (from proton to insensitive nuclei) must be set up. Usually it is accomplished by cross-polarization, while in highly mobile systems it may be obtained by INEPT, as in liquids. Several pulse sequences are routinely available for assignment and structural characterization, and local and global dynamics may be easily estimated.

The spectrometer provides time-domain data that are transformed automatically by the software into frequency-domain data.

Technical Specifications

 

Samples are packed in the rotors and excess water should be removed to avoid damage to both the sample and the probehead. The samples should not contain salts above 100 millimolar concentration. The measurement consists of the following steps:

  1. The spinning of the sample is started and the outer temperature is lowered accordingly to keep the temperature inside the rotor at a safe value.
  2. When spinning and temperature are stable, the resonance frequency of the probehead circuitry is tuned and matched for optimal pulsing and receiving. Pulses are calibrated both by direct excitation and cross-polarization.
  3. Cross-polarization (1H to X) and double cross polarization (from 1H to Y and from Y to X) parameters are optimized.
  4. Multidimensional spectra are recorded.