Emxnano benchtop spectrometer

A Bruker EMXnano benchtop spectrometer operating at the X-band frequency was used to conduct continuous wave ESR measurements at room temperature. A 20–40 μL sample was used in a micropipette (BRAND, Germany, with a diameter of 0.68, 0.86, or 1.2 mm). The CW ESR spectra were acquired with 100 kHz modulation frequency, 0.6–2 mW microwave power, 0.15 mT modulation amplitude, and 18 mT sweep width. In situ samples were subjected to signal averaging over 40 scans, while protein samples were averaged over 50 to 150 scans.

CW EPR spectroscopy was performed with singly labeled RAC at X-band (9.645 GHz) frequency in aqueous solution (RAC buffer: 40 mM HEPES pH 7.4, 100 mM KAc, 5 mM MgCl₂) at room temperature. Measurement parameters were adjusted such that the spectral line shape was not distorted by overmodulation or saturation effects. Typical settings on the used EMXnano benchtop spectrometer (Bruker Biospin) were a power of 3.162 mW, a modulation amplitude of 0.8 G at a modulation frequency of 100 kHz. Samples were loaded into capillary pipettes ringcaps with 1 mm inner diameter (Hirschmann), and sealed with Hemato-Seal capillary tube sealant (Fisherbrand). The magnetic field-axis was recalculated to the microwave-frequency of 9.6355 GHz, and the spectra were normalized to the maximum amplitude of the center field peak. The intensity low field peak ratio (LFPR) = I (3415.3 G)/I (3404.6 G) was calculated. LFPR were normalized to the LFPR of the protein spectrum in solution for each RAC variant. Error bars indicate the noise-low field peak ratio. Since concentration and thus signal-to-noise ratios were worse for spectra in presence of ribosomes, they were Savitzky–Golay filtered with an order of 2 and a frame of 101 for better illustration.