Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Measurement temperatures ranged from 5 to 30 K, using liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker). EPR samples were frozen and stored in liquid nitrogen. The EPR spectra shown are representative signals from at least two individual experiments. Spin quantification was performed through double integration of the EPR spectra and calculated relative to NrdB∆169Mn. Unless otherwise stated, all spectra were recorded at 10 K, microwave power 1 mW, frequency 9.28 GHz, modulation amplitude 10 G and modulation frequency 100 kHz.
Continuous flow cryostat
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Continuous Flow Cryostat is a laboratory equipment designed for low-temperature research. It provides a stable and controlled low-temperature environment for various applications. The core function of the cryostat is to maintain samples or specimens at precisely regulated temperatures, enabling researchers to conduct experiments and analyses in a cryogenic setting.
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Lab products found in correlation
Itc 503 temperature controller, by Oxford Instruments (9 mentions)
Xepr software package, by Bruker (6 mentions)
Elexys e500 spectrometer, by Bruker (6 mentions)
Er049x superx microwave bridge, by Bruker (6 mentions)
Shq0601 cavity, by Oxford Instruments (4 mentions)
Elexys 500x band spectrometer, by Bruker (2 mentions)
Te011 er 4122shqe cavity, by Oxford Instruments (2 mentions)
Emxmicro, by Bruker (1 mentions)
Esp 300 spectrometer, by Oxford Instruments (1 mentions)
Er 4116dm, by Bruker (1 mentions)
Matlab, by MathWorks (1 mentions)
Ifs 66v s ftir spectrometer, by Bruker (1 mentions)
Elexys e 500, by Bruker (1 mentions)
Superconducting magnet, by Oxford Instruments (1 mentions)
14 protocols using continuous flow cryostat
1
Characterization of Mn-Containing Proteins
2
EPR Characterization of HydA1 Enzyme
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The EPR spectra shown are representative signals from at least two individual experiments. The individual experiments show some preparation dependent differences, but the amplitude of these background signals are negligible compared to the signal intensity of the [2Fe]adt activated HydA1. Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity (Fig. 2 ) or a Bruker EMX micro equipped with an EMX Premium bridge and an ER4119 HS resonator (Fig. 5 and S13–S15† ), both equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Measurement temperatures ranged from 10 to 20 K, using liquid helium as coolant, with the following EPR settings unless otherwise stated: microwave power 1 mW, modulation amplitude 1 mT, modulation frequency 100 kHz. The spectrometer was controlled by the Xepr software package (Bruker).
3
Anaerobic Sample Preparation for EPR
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EPR samples were prepared under strict anaerobic conditions. The proteins were reduced with a 10-fold molar excess of sodium dithionite, and the reaction was monitored by UV-visible spectroscopy. The samples were transferred into quartz EPR tubes capped with rubber septa and immediately flash-frozen outside the glovebox. The EPR samples were stored in liquid nitrogen until further usage.
The CW EPR measurements were carried out on a Bruker Elexys 500X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator, equipped with an Oxford Instruments continuous-flow cryostat and an ITC 503 temperature controller (Oxford Instruments). Low temperatures were achieved using liquid helium as the coolant. The spectrometer was controlled by the Xepr software package (Bruker). Standard measuring parameters were 10-G modulation amplitude and 100-kHz modulation frequency. The spectra were averaged over either four or eight scans.
The CW EPR measurements were carried out on a Bruker Elexys 500X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator, equipped with an Oxford Instruments continuous-flow cryostat and an ITC 503 temperature controller (Oxford Instruments). Low temperatures were achieved using liquid helium as the coolant. The spectrometer was controlled by the Xepr software package (Bruker). Standard measuring parameters were 10-G modulation amplitude and 100-kHz modulation frequency. The spectra were averaged over either four or eight scans.
4
EPR Spectroscopy with Cryostat Setup
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Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). The Xepr software package (Bruker) was used for data acquisition and processing of spectra.
5
EPR Spectroscopic Analysis of Iron-Sulfur Clusters in Huc
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The composition of the iron–sulfur clusters of Huc was analysed by EPR spectroscopy. X-band EPR measurements were performed on a Bruker ELEXYS E500 spectrometer equipped with a SuperX EPR049 microwave bridge and a cylindrical TE011 ER 4122SHQE cavity in connection with an Oxford Instruments continuous flow cryostat. Measuring temperatures were achieved using liquid helium flow through an ITC 503 temperature controller (Oxford Instruments). Samples of Huc isolated in air were prepared under a neat argon atmosphere or flash-frozen following incubation under 1 atm H2, and X-band EPR spectra were collected in the temperature range 40–7 K, at varying microwave powers. Preliminary simulations of the Ni signals in the EPR spectra were carried out using EasySpin v.6.0.0-dev47 (ref. 78 (link)).
6
Q-band DEER Spectroscopy for Distance Distributions
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The dipolar time evolution data was obtained at 83–200 K using a standard DEER four-pulse protocol, (π/2)mw1–τ1–(π)mw1–τ1–(π)mw2–τ2–(π)mw1–τ2–echo,86 on a Bruker 580 pulsed EPR spectrometer operating at Q-band frequency (33.9 GHz). All pulses were square pulses with lengths of 12, 24, and 40 ns, for (π/2)mw1, (π)mw1, and (π)mw2, respectively. Temperature control was provided by an Oxford Instruments continuous flow cryostat controlled by an ITC503 temperature controller unit. Distance distributions were obtained from the time evolution data by assuming that the distance distribution can be approximated by a sum of Gaussians,87 ,88 (link) including Gaussian distance distributions with 95% confidence bands.65
7
EPR Spectroscopy of Cryogenic Samples
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Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments, Oxford, United Kingdom). Measurement temperatures ranged from 5 to 32 K, using liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker).
8
Continuous Wave EPR at Variable Temperatures
9
EPR Spectroscopy of Iron-Sulfur Proteins
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EPR samples (generally at 200 μM protein concentration) were prepared under strict anaerobic conditions. The proteins were reduced with 10–20 fold molar equivalents (2–4 mM) of NaDT or oxidized with 10–20 fold molar equivalents (2–4 mM) of thionine acetate for 20–40 minutes. The reduction was followed by UV/Vis, monitoring the disappearance of the [4Fe4S]2+ absorbance around 410 nm. The samples were transferred into quartz EPR tubes, capped with rubber septa before they were removed from the glovebox and immediately flash frozen. The EPR samples were stored in liquid nitrogen.
The low temperature CW EPR measurements were carried out with a Bruker Elexys 500 X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Low temperature measurements were carried out with liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker). Spectra were recorded with a 10 G modulation amplitude and a 100 kHz modulation frequency. Spectra were averaged over either four or eight scans.
The low temperature CW EPR measurements were carried out with a Bruker Elexys 500 X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Low temperature measurements were carried out with liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker). Spectra were recorded with a 10 G modulation amplitude and a 100 kHz modulation frequency. Spectra were averaged over either four or eight scans.
10
Characterization of Metal Complexes
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Nuclear magnet resonance measurements were recorded with a JEOL (400 YH magnet) Resonance 400 MHz. Infrared spectrum was collected with a Bruker IFS 66 v/S FT-IR spectrometer, in which the DLaTGS detector was employed with 2 cm -1 resolution. Sample solution was prepared inside a gastight IR cell with CaF 2 or KBr as windows. For UV-visible spectrum, the Carry 5000 spectrometer was used and the sample solution was placed in a gas-tight UV-visible cuvette. The mass spectrum was collected in a negative mode with a Thermo Finnigan LCQ Deca XP Max LC/MS spectrometer through the direct injection mode. EPR spectra were recorded at X-band at 9.28 GHz, modulation frequency 100 kHz and modulation amplitude 5 Gauss on a Bruker ELEXYS E500 using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and an ITC 503 temperature controller (Oxford Instruments). A 1 mM CuSO 4 and 10 eq. of EDTA dissolved in water was employed as a standard sample for spin counting.
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