Overview of the system architecture for cryogenic MAS experiments and sample exchange. Thick black lines indicate vacuum-jacketed transfer lines.

CAD drawings of the probe instrumentation needed for cryogenic MAS. a) Evacuated bearing and drive transfer lines in the probe deliver the cryogens to the probe, while an exhaust transfer line brings the cold cryogens out of the probe without cooling the box containing the tuning elements. 50 W heaters at the termination of the flexible evacuated transfer lines allow for precise control of the temperature. The probe-box is purged with a low pressure flow of room temperature nitrogen gas to prevent condensation. b) Cut-out of probe. Multiple optical channels are required for in situ optical irradiation of the sample, temperature sensing, and monitoring of the MAS frequency. Modifications to the dewar, an angled eject pipe that mates to a commercially available Revolution NMR stator, a GORE-TEX seal, and other modifications are needed for the cryogenic sample exchange system.

Sample ejection schematics a) Cut-away of probe showing the rotor (purple). A burst of nitrogen gas into the exhaust port creates a flow of gas out of the probe and into the vacuum-jacketed sample exchange tube, carrying the rotor from the stator (1) into the angled sample eject pipe (1 and 2) and finally into the vertical exchange tube (4). For sample insertion, the rotor is lowered on a cushion of gas from the top of magnet and follows the reverse path. b) Angled sample eject pipe. The rotor exits the stator and is guided by the bottom surface of the eject pipe to make the turn from 54.5° to 0° with respect to the magnet bore axis in the limited space provided. The top of the eject pipe is a cone which fits inside the larger cone of the vertical eject tube to allow for adjustment of the magic angle. c) Vertical sample exchange tube. A threaded aluminum attachment allows for a secure coupling into the probe-dewar and proper alignment of tube with respect to the cone on the sample eject pipe. A custom designed valve is closed during operation to prevent frost formation, and is remotely opened during sample exchange. For ejection the rotor travels into a ½″ Teflon tube which coils around to slowly and safely slow the rotor.

a) RF circuit diagram. The sample coil and capacitor at the probe (for ¹⁵N isolation) are connected to the box via a transmission line (T_main). b) Computer assisted design rendition of the RF circuit elements. Photographs of the actual RF circuit are shown in the appendix.

Chromophore in the active site of bacteriorhodopsin with a covalent Schiff base linkage to Lys216. The retinylidene assumes a 13-cis, 15-syn conformation in bR₅₅₅ and 13-trans, 15-anti in bR₅₆₈. These conformers are named from the absorption maximum in the UV-VIS spectrum; at 568 nm for bR₅₆₈ and 555 nm in bR₅₅₅.

RFDR spectrum of 14, ε-¹³C bacteriorhodopsin: 15 mg of protein in 60 vol% d₈-glycerol:40 vol% aqueous 0.3 M Guanadine HCl (pH = 10). ω_r/2π = 8000 Hz, temperature is 93 K at the stator, 32 indirect T₁ points, spectrum recorded in 7.4 hours.

Heat exchanger. The pressure of the inner can is regulated and controls the output temperature of the cryogen. The nitrogen reservoir is replenished automatically during operation by means of a level sensor and electromechanical valve.

Thermal isolation strategies a) vacuum-jacketed transfer lines used to deliver the bearing and drive cryogens and transport the exhaust gas out of the probe. b) non-magnetic probe dewar with a bellowed transfer pathway to accommodate the sample ejection pipe (from the bottom) and transfer tube (from the top).

250 GHz microwave channel and rotor details. a) Enlarged view of the probe with selected components shown. A small groove in the vespel drive-tip is filled with a cryogenic epoxy, Hysol EA 9361 (Loctite) to prevent the drive-tip from coming loose at cryogenic temperatures. The epoxy is also used to seal the top of the rotor. The top Kel-F spacer is threaded, providing a simple and safe way to empty and fill the rotor; the epoxy can be removed with a standard epoxy-stripper, while a 0-80 threaded tool threads into the top spacer, providing a grip to pull the spacer out of the rotor. The microwave power is coupled from the HE₁₁ mode of the corrugated waveguide to the sample by launching the microwaves from the waveguide in the form of a free space propagating Gaussian beam. b) a system of mirrors inside the probe-box focuses the beam delivered by the horizontal corrugated waveguide from the gyrotron onto the vertical waveguide leading to the sample. The red lines trace the trajectory of the microwaves.

Spectra acquired with the cryogenic sample exchange equipped DNP probe. Asterisks identify spinning sidebands. a) Stack-plot showing stable enhancements of 1 M ¹³C-urea between sample ejections. The matrix consists of, by volume, 60% d₈-glycerol, 30% H₂O, 10% D₂O, and 15 mM TOTAPOL [19]. The lower three spectra were collected without MW radiation, and the upper three with 250 GHz microwave irradiation, enabling ∼ 100-fold DNP enhancement. The spinning frequency was 4000 +/- 5 Hz and the temperature in the probe was 85 K for all three trials. b) ¹³C spectrum of natural abundance N-formyl-Met-Leu-Phe-OH (f-MLF-OH) recorded at 100 K. The linewidth at half-height of three resonances is indicated. ω_r/2π = 6000 Hz. 83 KHz ¹H TPPM [20] decoupling was applied for the 50.8 ms of acquisition,. No line-broadening or linear prediction was used. The recycle delay was 3.5 seconds, 2048 transients were averaged. f-MLF-OH was obtained from Bachem (King of Prussia, PA).

Extraction of C14-Cε distance in the active site of bacteriorhodopsin. a) Least-squares fits of RFDR data yielding a distance of 3.90 Å between C14 and Cε in bR₅₆₈ (red) and 3.11 Å in bR₅₅₅ (black). The seven mixing points took a total of 52 hours to record. b) Residual sum of squares surface yielding a 95% confidence interval of 3.90 +/- 0.08 Å, and 11.7 ms +/- 2.6 ms for bR₅₆₈. c) Similar contour plot for bR₅₅₅, yielding 3.11 +/- 0.22 Å, and 54.7 ms +/- 19.2 ms for T_2ZQ 95% confidence intervals.

a) Photograph of the probe. b) The inside of the probe box showing the RF circuit. c) Top of the probe-box showing the seals to the probe-dewar. d) Mirrors on the bottom plate of the probe-box for coupling microwaves from the horizontal waveguide to the vertical waveguide e) Sample insertion chamber at the top of the magnet f) Deceleration tube that gently stops the rotor during the ejection process.