CW-EPR and ST-EPR experiments were performed on a Varian E-109 spectrometer fitted with a two-loop one-gap resonator [106 (link), 107 (link)]. SR-EPR and Four-pulse DEER experiments were conducted on the Bruker ELEXSYS 580 spectrometer. Spectral simulations and DEER data analyses were carried out using the programs “MultiComponent” and “LongDistances”, respectively. The programs are available at https://www.biochemistry.ucla.edu/Faculty/Hubbell/software.html . Additional details in SI Materials and Methods.
Elexsys 580 spectrometer
Manufactured by Bruker
The Elexsys 580 is a high-performance electron paramagnetic resonance (EPR) spectrometer designed for advanced research applications. It provides precise measurement and analysis of the magnetic properties of materials with unpaired electrons.
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6 protocols using elexsys 580 spectrometer
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EPR Spectroscopy Experimental Protocols
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EPR Spectrometry of Aqueous Mn(II) Samples
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Electron paramagnetic resonance (EPR) spectra were acquired with a Bruker Elexsys 580 spectrometer fitted with an SHQE resonator and a Bruker continuous flow nitrogen VT insert. Room temperature aqueous samples were contained in 1.0 mm OD × 0.8 mm ID quartz tubes (Vitrocom) sealed with Critoseal capillary tube sealant (Leica Microsystems). These were placed in conventional 4 mm OD × 3 mm ID quartz EPR tubes (Wilmad). The microwave frequency was ~9.86 GHz (~9.33 GHz without the VT insert), and microwave power was 20 mW. The field was swept at 1000 Gauss (G) in 84 s and modulated at a frequency of 100 kHz with 5 G amplitude. A time constant of 82 ms was employed, and typically 20 scans were averaged. Quantitation of Mn(II) reprised the method of Reardon et al.23 (link).
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Pulsed EPR Measurements of Protein Structures
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Pulsed EPR measurements were performed at Q band (34 GHz) and −223 °C on an Elexsys 580 spectrometer (Bruker). Therefore, 15 μl of the freshly prepared samples were loaded into EPR quartz tubes with a 1.6 mm outer diameter and shock frozen in liquid nitrogen. During the measurements, the temperature was controlled by the combination of a continuous-flow helium cryostat (Oxford Instruments) and a temperature controller (Oxford Instruments). The four-pulse DEER sequence was applied64 (link) with observer pulses of 32 ns and a pump pulse of 13–18 ns. The frequency separation was set to 70 MHz and the frequency of the pump pulse to the maximum of the nitroxide EPR spectrum. Validation of the distance distributions was performed by means of the validation tool included in DeerAnalysis65 (link) and varying the parameters “Background start” and “Background density” in the suggested range by applying fine grid. A prune level of 1.15 was used to exclude poor fits. Furthermore, interspin distance predictions were carried out by using the rotamer library approach included in the MMM software package19 (link). The calculation of the interspin distance distributions is based on the cryo-EM structures of state 1, state 2 and the crystal structure [5MRW]4 (link) for the comparison with the experimentally determined interspin distance distributions.
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EPR Spectroscopy of Frozen and Liquid Samples
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Decoherence Dynamics of Phosphorus Donors in Silicon
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Experimental results were measured on a natural silicon Czochralski wafer doped with 3 ×
1014 per cm3 phosphorus, using an X-band
(9.6 GHz) Bruker ELEXSYS 580 spectrometer. All decay times were
obtained on the high-field electron spin resonance line
(mI=−1/2) at 3,452 G at
6 K (where the electron spin relaxation processes
(T1≈1 s) did not contribute to
decoherence over the timescales considered in this paper). The multiple pulses
required for the DD sequences can result in ‘stimulated
echoes’, and other unwanted echoes, in the experiment due to pulse
infidelities. When such echoes overlap with the desired one (from spin packets
which have been flipped by all the π pulses), the
experimentally observed decay curves gain unwanted contributions. We therefore
cycled the phases of the applied π pulses in such a way as to
remove the contribution of all undesired echoes. For UDD, the timings between
each pulse are different and most stimulated echoes fall outside the desired one
which can then be isolated. For example, the phase cycling sequence for UDD-4
requires simply subtracting the echo from two experiments where the first two
pulses are changed from +π to –πand the last two are +π. For CMPG, this is more challenging as
the intervals are equal and we did not suppress all possible stimulated echoes
for CPMG-5 and CPMG-6.
1014 per cm3 phosphorus, using an X-band
(9.6 GHz) Bruker ELEXSYS 580 spectrometer. All decay times were
obtained on the high-field electron spin resonance line
(mI=−1/2) at 3,452 G at
6 K (where the electron spin relaxation processes
(T1≈1 s) did not contribute to
decoherence over the timescales considered in this paper). The multiple pulses
required for the DD sequences can result in ‘stimulated
echoes’, and other unwanted echoes, in the experiment due to pulse
infidelities. When such echoes overlap with the desired one (from spin packets
which have been flipped by all the π pulses), the
experimentally observed decay curves gain unwanted contributions. We therefore
cycled the phases of the applied π pulses in such a way as to
remove the contribution of all undesired echoes. For UDD, the timings between
each pulse are different and most stimulated echoes fall outside the desired one
which can then be isolated. For example, the phase cycling sequence for UDD-4
requires simply subtracting the echo from two experiments where the first two
pulses are changed from +π to –πand the last two are +π. For CMPG, this is more challenging as
the intervals are equal and we did not suppress all possible stimulated echoes
for CPMG-5 and CPMG-6.
6
X-band HYSCORE Spectroscopy for Hyperfine Tensor Determination
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