Cf935

EPR spectra were collected on an X-band CW EPR spectrometer (Bruker Biospin GmbH) using an X-band Super High Sensitivity Probehead (Bruker) and equipped with a low temperature helium flow cryostat (Oxford Instruments CF935). Data were analyzed using the program EasySpin (23 (link)).

For PELDOR measurements, 15–20 μl of cf-TMD0-His S₂₈C or S₁₃₈C sample containing 15–20% (v/v) deuterated glycerol was transferred into 1.6 mm outer diameter quartz EPR tubes (Suprasil, Wilmad LabGlass) and quick-frozen in liquid nitrogen. Pulsed EPR data were recorded on an ELEXSYS E580 EPR spectrometer (Bruker), which is equipped with a PELDOR unit (E580-400U, Bruker), a continuous-flow helium cryostat (CF935, Oxford Instruments), and a temperature control system (ITC 502, Oxford Instruments). Measurements were performed at Q-band frequencies (33.4 GHz) using an ELEXSYS SuperQ-FT accessory unit and a Bruker AmpQ 10 W amplifier in a Bruker EN5107D2 cavity at 50 K. The dead-time free four-pulse sequence with phase-cycled π/2-pulse was used for PELDOR measurements^{36 (link)}. A 20 ns pump pulse was used, which was placed at the maximum of the echo-detected field swept spectrum. The observer pulse lengths were set to 32 ns (π/2 and π), which were set 70 MHz lower. The deuterium modulations were averaged by increasing the first interpulse delay by 16 ns for 8 steps. The normalized primary PELDOR data V(t)/V(0) were processed to remove background contribution, and the resulting form factors F(t)/F(0) were fitted with a model-free Tikhonov regularization to distance distributions with DeerAnalysis2016 software package^{37 (link)}.

PELDOR experiments were performed at 50 K on a Bruker ELEXSYS E580 spectrometer operating at 9.6 GHz equipped with an ER 4118 X-MD5 resonator, Oxford Instruments continuous flow cryostat (CF935) and ITC503 temperature controller. The 4-pulse PEDLOR sequence used was π/2(ν_obs) − τ₁ − π(ν_obs) − t′ − π(ν_pump) − (τ_l + τ₂ − t′) − π(ν_obs) − τ₂ − echo, where the π/2 observer pulse length was 16 ns. The pump pulse length was 12 ns. τ₁ was 200 ns, while the long interpulse delay (τ₂) was 2,000 ns. The PELDOR time traces have a resolution of 8 ns. Each scan includes phase cycling to remove the receiver offset, and nuclear modulation averaging to remove residual ESEEM (Electron Spin Echo Envelope Modulation) contributions. All data are the average of nine scans. For each measurement, the microwave channels’ amplitudes were optimized, whereas the receiver video gain and all other parameters were fixed.
The time traces were analyzed using the program DeerAnalysis 2013 [28 (link)]. The background was corrected by a homology three-dimensional fit and the distance distributions were evaluated according to worm-like chain (WLC) model [28 (link)] as previously reported for this model system [27 (link)].

All pulsed EPR experiments were implemented on an X-band Bruker ELEXSYS E580 with a Bruker EN 4118-X-MD4 pulse ENDOR resonator. The temperature was maintained with a helium cryostat (Oxford Instruments CF935). For experiments at cryogenic temperatures, the samples were flash frozen using liquid nitrogen and then inserted into the cryostat. Field swept echo detected EPR spectra were acquired using a standard two-pulse Hahn echo sequence with $\pi /2$ pulse length of 16 ns, $\pi$ pulse length of 32 ns and an inter-pulse delay $\tau$ of 200 ns. For $T_1$ measurements, an inversion recovery sequence ( $\pi$ – T – $\pi /2$ – $\tau$ – $\pi$ – $\tau$ – echo) with 4-step phase cycle was used. A $\pi /2$ pulse length of 14 ns and $\pi$ pulse lengths of 28 ns were used. $\tau$ was kept at 200 ns. The delay T after inversion was set to an initial value of 400 ns and incremented linearly in steps of 2  $\mathrm{\mu }\text{s}$ to obtain a data set with 1024 recovery times. Laplace inversion of $T_1$ relaxation data was done using an exponential kernel without non-negativity constraint, with pre-processing of data and parametrization as described elsewhere.^{35 (link),44 (link)} The inversion was performed using home-written scripts that were run on Octave v. 6.4.