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1. PURPOSE
This large sample 15N-1H probe was designed for solid state NMR structure determination of oriented membrane proteins.
It is used for PISEMA (polarization inversion spin exchange at magic angle)
experiments to obtain orientational restraints from membrane proteins in a lipid environment.
The probe is used in tandem with console-programmable upper VT stack, which was also built
in-house to free up valuable physical space inside the probe for high-voltage components.
In general, principal challenges in building probes for solid state NMR of membrane proteins are:
- Large sample size with RF coils exceeding 1/4 wavelength
- High B1 homogeneity requirement for proton channel
- High efficiency of both channels, especially of 15N channel
- High power handling capability on both channels
- Accoustic ringing at 15N frequency
- Sample preparation and packaging that optimises filling factor while maintaining hydration for long time
- Sample heating resulting from high power decoupling pulse
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| probehead view |
2. PROBE FEATURES
- Large RF coil: >600 uL, 8 x 6 x 12 mm coil size
- Balanced proton RF channel for improved B1 homogeneity:
- A(810°)/A(90°) = 71% for proton
- A(810°)/A(90°) = 76% for nitrogen
- 90° pulse lengths:
- 2.3 us at 300 W for proton (109 kHz)
- 4.7 us at 670 W for nitrogen (53 kHz)
- Rated input power: 350W at 1H, 900W at 15N
- Channel isolation is better than -25dB both ways
- Acoustic ringing time in nitrogen channel is less than 15 us.
- Temperature chamber range: -40°C...+100°C
- Temperature regulation delivered by upper VT stack
3. EXAMPLE
2D PISEMA spectra of 8mg 15N-Leu26,36,38,40,43-M2-TMP oriented in DMPC in the presence of 10 mM Amantadine. A recycle delay of 6 seconds was employed.
4. B1 HOMOGENEITY
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B1 homogeneity test of proton channel.
A(810°)/A(90°) = 71% |
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B1 homogeneity test of nitrogen channel.
A(810°)/A(90°) = 76% |
Two figures above show B1 homogeneity test for both proton and nitrogen frequency.
Good RF homogeneity inside RF coil is important for uniform cross-polarization during the experiment.
These tests were performed on standard sample with dimensions of 6 x 4 x 9 mm.
Moving the sample by 2 mm in either direction along the axis resulted in no change of A(810°) to A(90°) ratio.
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